Protein Interactions in Retina Development and Function

视网膜发育和功能中的蛋白质相互作用

• 批准号: 6826697
• 负责人: SOFIA P BECERRA
• 金额: --
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6826697
• 关键词: Primates; binding sites; biological signal transduction; cell differentiation; confocal scanning microscopy; enzyme activity; enzyme inhibitors; extracellular matrix proteins; fluorimetry; growth factor receptors; heparan sulfate; histogenesis; hypoxia; laboratory rat; motor neurons; neural degeneration; neurotrophic factors; polymerase chain reaction; protease inhibitor; protein protein interaction; protein structure function; receptor binding; retinal pigment epithelium; serine proteinases; tissue /cell culture; western blottings

Work in this research group is aimed at elucidating the mechanisms that regulate the activites of pigment epithelium-derived factor (PEDF), an extracellular neurotrophic and antiangiogenic protein. PEDF induces neuronal differentiation on retinoblastoma Y-79 cells via interactions with cell-surface receptors in these cells. It also has binding affinity for glycosaminoglycans. We investigated the effects of glycosaminoglycans on PEDF-receptor interactions. Using specific glycosaminoglycan degrading enzymes in spectrophotometric assays and PEDF-affinity chromatography, we detected heparin and heparan sulfate-like glycosaminoglycans in the Y-79 conditioned media, which had binding affinity for PEDF. The Y-79 conditioned media significantly enhanced the binding of 125I-PEDF to Y-79 cell-surface receptors. However, enzymatic and chemical depletion of sulfated glycosaminoglycans from the Y-79 cell cultures by heparitinase and chlorate treatments decreased the degree of 125I-PEDF binding to cell-surface receptors. Thus heparin/heparan sulfate may be important in regulating the activity of PEDF by providing efficient cell-surface receptor binding. To investigate the effects of hypoxia on PEDF, we exposed retina pigment epithelial (RPE) cells to low oxygen or to chemical agents that mimic hypoxia. Hypoxia reduced the PEDF levels in the RPE media, but its gene expression remained unchanged compared to normoxic controls. Hypoxia induced gelatinolytic activities in the RPE media, which were EDTA-sensitive and calcium-dependent, degraded PEDF, and immunoreacted with MMP-2 antibodies. Vitreous and BHK[pPEDF] cell conditioned media also contained these activities. Purified MMP-2 and MMP-2 induced in BHK[pPEDF] cells by VEGF were able to specifically and completely degrade PEDF. Thus hypoxia can downregulate PEDF expression at a post-translational level by MMP-2 proteolytic degradation. We also investigated the effects of dexamethasone on PEDF in collaboration with the laboratory of Dr. T. Borras (UNC). Dexamethasone was used to perfuse anterior segment organ cultures prepared from paired eyes from post-mortem human donors and treat cultured cells from human trabecular meshwork (HTM). Dexamethasone increased the PEDF mRNA of trabecular meshwork from perfused segments over contralateral controls and of cultured HTM cells over vehicle-treated cells. It also increased the levels of secreted PEDF protein in effluents of organ cultures and in HTM cell media above controls. Confocal microscopy of HTM tissue showed a positive correlation between increased PEDF immunofluorescence and dexamethasone-perfusion. Thus dexamethasone can upregulate PEDF expression at a transcriptional level. To establish novel routes of protein delivery to the retina, we evaluated the diffusion of fluorescein-conjugated proteins, PEDF (50-kDa) and ovalbumin (45-kDa), through monolayers of monkey RPE cells and pig sclera tissue explants, and in rat eyes in vivo. Transepithelial resistance and voltage, as well as the lack of trypan blue and horseradish peroxidase diffusion confirmed confluency and tight junction formation of the cultured RPE cells. Fl-PEDF passed through the RPE monolayers from either the apical or basal side, as determined by immunoblotting, fluorometry, and confocal microscopy. Fl-ovalbumin diffused through the scleral tissue at a constant ratio, as determined by fluorometry (in collaboration with the laboratory of R. Lutz, OD/ORS). In animal models injected subconjunctivally with Fl-PEDF or with Fl-ovalbumin implants, microscopy revealed fluorescein diffusion into the choroid/RPE complex and into the photoreceptor outer segments suggesting that subconjunctival protein delivery can be a means for increasing the PEDF levels in the retina.

该研究组的工作旨在阐明调节色素上皮衍生因子（PEDF）的机制，一种细胞外神经营养和抗血管生成蛋白。 PEDF通过与这些细胞中细胞表面受体的相互作用在视网膜母细胞瘤Y-79细胞上诱导神经元分化。它还对糖胺聚糖具有结合亲和力。我们研究了糖胺聚糖对PEDF受体相互作用的影响。我们使用分光光度测定和PEDF亲密色谱中的特定糖胺聚糖降解酶，我们在Y-79条件培养基中检测到肝素和硫酸乙酰肝素样的硫酸盐样糖胺聚糖，这些培养基具有PEDF的结合亲和力。 Y-79条件培养基显着增强了125i-PEDF与Y-79细胞表面受体的结合。然而，肝素酶和氯酸盐治疗从Y-79细胞培养物中硫化糖胺聚糖的酶促和化学耗竭降低了125i-PEDF与细胞表面受体的结合程度。因此，通过提供有效的细胞表面受体结合来调节PEDF的活性可能很重要。 为了研究缺氧对PEDF的影响，我们将视网膜色素上皮（RPE）细胞暴露于低氧或模仿缺氧的化学剂中。缺氧降低了RPE培养基中的PEDF水平，但与常氧对照相比，其基因表达保持不变。缺氧诱导RPE培养基中的明胶活性，这些培养基对EDTA敏感和钙依赖性，降解PEDF，并用MMP-2抗体进行免疫反应。玻璃体和BHK [PPEDF]细胞调节培养基也包含这些活性。 VEGF在BHK [PPEDF]细胞中诱导的纯化的MMP-2和MMP-2能够具体并完全降解PEDF。因此，缺氧可以通过MMP-2蛋白水解降解在翻译后水平下下调PEDF表达。我们还与T. Borras博士（UNC）合作研究了地塞米松对PEDF的影响。地塞米松用于灌注由验尸后供体配对的眼睛制备的前节器官培养物，并从人类小梁网（HTM）中处理培养的细胞。地塞米松增加了小梁网的pEDF mRNA，而小梁网的对侧对照对侧对照和培养的HTM细胞在媒介物处理的细胞上的pEDF mRNA。它还增加了器官培养物和上述HTM细胞培养基中的废水中分泌的PEDF蛋白水平。 HTM组织的共聚焦显微镜表现出pEDF免疫荧光增加与地塞米松 - 灌注之间的正相关。因此，地塞米松可以在转录水平上上调PEDF表达。 为了建立新的蛋白质递送到视网膜的途径，我们通过猴子RPE细胞和猪sclera Tissue Epplints的单层以及在Vivo中评估了荧光素偶联蛋白，PEDF（50-kDa）和卵形蛋白（45 kDa）的扩散。旋转的电阻和电压，以及缺乏锥虫蓝和辣根过氧化物酶扩散证实了培养的RPE细胞的汇合和紧密的结。通过免疫印迹，荧光测定法和共聚焦显微镜确定，FL-PEDF从顶端或基础侧穿过RPE单层。 Fl-overbumin通过荧光测定法确定（与R. Lutz，OD/ORS的实验室）确定，以恒定比率扩散。在动物模型中，向fl-PEDF或fl-overbumin植入物注入了亚缝合，显微镜显示荧光素扩散到脉络膜/RPE复合物中以及光感受器的外部片段，这表明亚临时蛋白质递送可能是增加视网膜中PEDF水平的一种手段。