Molecular Mechanisms Of Glaucoma

青光眼的分子机制

基本信息

批准号：
7734607
负责人：
Stanislav I Tomarev
金额：
$ 106.28万
依托单位：
NATIONAL EYE INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

It is now well established that a genetic component may contribute to glaucoma, and several glaucoma-associated genes have been identified. The first identified and the most studied gene is Myocilin, which is heavily expressed in and secreted by the trabecular meshwork, one of the key components of the eye aqueous humor outflow system. The myocilin protein belongs to a family of glycosylated proteins containing a C-terminal olfactomedin domain. Dominant mutations in Myocilin are found in 3-4% of patients with primary open angle glaucoma and most of these glaucoma-causing mutations are located in the olfactomedin domain. The function of wild-type Myocilin is not clear. To study functions of human myocilin, wild-type and mutated myocilin were expressed in HEK293 cells under inducible promoter. Conditioned medium from myocilin-expressing cells as well as purified myocilin induced formation of stress fibers in primary cultures of human trabecular meshwork or NIH3T3 cells. Stress fiber-inducing activity of myocilin was blocked by antibodies against myocilin as well as secreted inhibitors of Wnt-signaling, sFRP1 or sFRP3. Interaction of myocilin with sFRP3 and several frizzled receptors was confirmed by immunoprecipitation experiments and by binding of myocilin to the surface of cells expressing cysteine-rich domains of different sFRPs and Frizzled proteins. Treatment of NIH3T3 cells with myocilin and its proteolytic fragments induced intracellular re-distribution of -catenin and its accumulation on the cellular membrane but did not induce nuclear accumulation of -catenin. Overexpression of myocilin in the eye angle tissues of transgenic mice stimulated accumulation of -catenin in these tissues. Myocilin and Wnt proteins may perform redundant functions in the mammalian eye, as myocilin modulates Wnt signaling by interacting with components of this signaling pathway.

现在已经很好地确定，遗传成分可能有助于青光眼，并且已经鉴定出了几种与青光眼相关的基因。第一个鉴定出和研究最多的基因是肌动蛋白，该基因在小梁网中大量表达并分泌，这是眼睛水性幽默流出系统的关键组成部分之一。肌动蛋白蛋白属于含有C-末端嗅觉蛋白结构域的糖基化蛋白家族。在3-4％的原发性开角青光眼患者中发现了肌动蛋白的显性突变，这些引起青光眼的突变大多数位于橄榄毒素结构域中。野生型肌动蛋白的功能尚不清楚。为了研究人肌动蛋白的功能，在诱导型启动子下，在HEK293细胞中表达了野生型和突变的肌动蛋白。来自表达肌钙蛋白的细胞的条件培养基以及纯化的肌动蛋白诱导的人小梁网或NIH3T3细胞的原代培养物的应激纤维形成。肌动蛋白的应激纤维诱导活性被针对肌动蛋白的抗体以及WNT信号，SFRP1或SFRP3的分泌抑制剂所阻断。通过免疫沉淀实验以及肌核酸与表达不同SFRP和毛线蛋白质富含半胱氨酸结构域的细胞表面结合来证实肌动蛋白与SFRP3和几个毛躁受体的相互作用。用肌动蛋白及其蛋白水解片段对NIH3T3细胞的治疗诱导的细胞内重新分布-Catenin及其在细胞膜上的积累，但并未诱导-Catenin的核积累。转基因小鼠的眼角组织中肌动蛋白的过表达刺激了这些组织中-Catenin的积累。肌动蛋白和Wnt蛋白可以在哺乳动物眼中执行冗余功能，因为肌动蛋白通过与该信号通路的组件相互作用来调节Wnt信号传导。