Locke Christina J, Congrove Nicole R, Dismuke W Michael, Bowen Trent J, Stamer W Daniel, McKay Brian S
Department of Ophthalmology and Vision Science, University of Arizona, Tucson, AZ, USA.
Department of Ophthalmology, Duke University, Durham, NC, USA.
Exp Eye Res. 2014 Dec;129:1-4. doi: 10.1016/j.exer.2014.10.010. Epub 2014 Oct 11.
Retinal Pigment Epithelial cells (RPE) express both GPR143 and myocilin, which interact in a signal transduction-dependent manner. In heterologous systems, activation of GPR143 with ligand causes transient recruitment of myocilin to internalized receptors, which appears to be the entry point of myocilin to the endocytic pathway. In some but not all cells, myocilin also traffics through the multivesicular body (MVB) and is released on the surface of exosomes in a signal transduction-dependent fashion. Little is known regarding the role of exosomes in RPE, but they likely serve as a mode of communication between the RPE and the outer retina. In this study, we used posterior poles with retina removed from fresh human donor eyes as a model to test the relationship between GPR143, myocilin, and exosomes in an endogenous system. We isolated exosomes released by RPE using differential centrifugation of media conditioned by the RPE for 25 min, and then characterized the exosomes using nanoparticle tracking to determine the number and size of the exosomes. Next, we tested whether ligand stimulation of GPR143 using l-DOPA altered RPE exosome release. Finally, we investigated whether myocilin was present on the exosomes released by RPE and whether l-DOPA stimulation of GPR143 caused recruitment of myocilin to the endocytic pathway, as we have previously observed using cultured cells. Activation of GPR143 halted RPE exosome release, while simultaneously recruiting myocilin to the endocytic compartment. Together, our results indicate that GPR143 and myocilin function in a signal transduction system that can control exosome release from RPE.
视网膜色素上皮细胞（RPE）表达GPR143和肌纤蛋白，二者以信号转导依赖的方式相互作用。在异源系统中，用配体激活GPR143会导致肌纤蛋白短暂募集到内化受体上，这似乎是肌纤蛋白进入内吞途径的切入点。在一些但并非所有细胞中，肌纤蛋白也会通过多囊泡体（MVB）运输，并以信号转导依赖的方式释放到外泌体表面。关于外泌体在RPE中的作用知之甚少，但它们可能是RPE与视网膜外层之间的一种通讯方式。在本研究中，我们使用从新鲜人类供体眼中移除视网膜的后极作为模型，来测试内源性系统中GPR143、肌纤蛋白和外泌体之间的关系。我们通过对RPE条件培养基进行25分钟的差速离心，分离出RPE释放的外泌体，然后使用纳米颗粒追踪技术对外泌体进行表征，以确定外泌体的数量和大小。接下来，我们测试了使用左旋多巴（l-DOPA）刺激GPR143是否会改变RPE外泌体的释放。最后，我们研究了RPE释放的外泌体上是否存在肌纤蛋白，以及GPR143的l-DOPA刺激是否会导致肌纤蛋白募集到内吞途径，正如我们之前在培养细胞中观察到的那样。GPR143的激活阻止了RPE外泌体的释放，同时将肌纤蛋白募集到内吞区室。我们的结果共同表明，GPR143和肌纤蛋白在一个可以控制RPE外泌体释放的信号转导系统中发挥作用。
