Signaling in the retina and retinal pigment epithelium

视网膜和视网膜色素上皮中的信号传导

• 批准号: 7594101
• 负责人: Thomas Redmond
• 金额: $ 190.82万
• 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/7594101
• 关键词: 11 cis Retinal; Adherence; Adherens Junction; Affect; Affinity Chromatography; Age related macular degeneration; All-Trans-Retinol; Amino Acids; Ankyrin Repeat; Antibodies; Apoptosis; Apoptotic; Area; Attention; Binding; Binding Proteins; Bos taurus; C-terminal; Cattle; Cell Death; Cell Line; Cell membrane; Cell physiology; Cells; Coiled-Coil Domain; Collaborations; Confocal Microscopy; DNA Damage; Diabetic Retinopathy; Dose; Epithelial; Epithelial Cells; Event; Excision; Exposure to; Family member; Fenretinide; Gene Family; Generations; Genes; Goals; Growth; Homeostasis; Human; IGFBP5 gene; Immunofluorescence Immunologic; Inflammatory; Insulin-Like Growth Factor Binding Protein 5; Insulin-Like Growth Factor I; Isomerism; Kinesin; Ligands; Light; Lipofuscin; Malignant Neoplasms; Mediating; Mediator of activation protein; Membrane; Metabolism; Monoclonal Antibodies; Mus; N-terminal; Neuronal Differentiation; Neurons; Nuclear; Nuclear Hormone Receptors; Numbers; Orthologous Gene; Oryctolagus cuniculus; Oxidative Stress; Pathology; Pathway interactions; Photoreceptors; Play; Polysaccharides; Preparation; Preventive; Process; Protein Analysis; Protein Isoforms; Proteins; RXR; Rate; Reactive Oxygen Species; Reagent; Regulation; Relative (related person); Retina; Retinal Diseases; Retinal Pigments; Retinitis Pigmentosa; Retinoids; Role; Sequence Analysis; Signal Transduction; Signal Transduction Pathway; Staining method; Stains; Stimulus; Stress; Structure; Structure of retinal pigment epithelium; Surface; System; Techniques; Tertiary Protein Structure; Therapeutic Agents; Time; Transcriptional Activation; Transcriptional Regulation; Tretinoin; Up-Regulation; Variant; Vitamin A; Western Blotting; Work; Yeasts; analog; base; biological adaptation to stress; caspase-2; cell growth; day; heme oxygenase-1; immunoreactivity; interstitial retinol-binding protein; monolayer; novel; oxidation; protein function; protein protein interaction; receptor; research study; response; retinamide; transcription factor; uptake; visual cycle; yeast two hybrid system

The RPE, a monolayer of highly differentiated epithelial cells located between the photoreceptors and choriocapillaries, is exposed to variety of stress, including exposure to light, inflammatory mediators, and reactive oxygen species. Apoptotic RPE cell death resulting from increased oxidative stress could hasten the onset of age-related macular degeneration (AMRD). Retinoic acid, derived from oxidation of vitamin A, affects many cellular functions including cell growth, differentiation, and apoptosis. This effect is mediated through transcriptional regulation by the nuclear hormone receptors RAR and RXR for which retinoic acids are ligands. Synthetic analogs of retinoic acid also have significant effects on cellular function. One such analog, fenretinide, N-(4-hydoxyphenyl)retinamide (4HPR), has long been used as a cancer preventive agent. Recently, it has been proposed as a therapeutic agent for lipofuscin-based retinal diseases. At low doses, we have shown that 4HPR induces neuronal differentiation of cultured ARPE-19 human retinal pigment epithelial cells. At higher doses it causes apoptosis. NORPEG (novel retinal pigment epithelial cell gene, RAI14), another retinoic acid regulated gene that we originally characterized from the human retinal pigment epithelial (RPE) cell line ARPE-19, encodes a protein consisting of 980 amino acid residues. The presence of ankyrin repeats region and coiled-coil domains, structural domains implicated in protein-protein interactions, suggests that NORPEG associates with multiple binding partners. It may be an important organizing protein in this regard. While most, if not all, enzymatic or binding protein components of the visual cycle have been identified, signaling events in the visual cycle have received less attention. It is anticipated that visual cycle retinoid flux is regulated by such external stimuli as day/night status, ambient light level, as well as by relative levels of retinoid isomers. Receptor mediated uptake of all-trans retinol as well as secretion of 11-cis retinal, both perhaps involving interphotoreceptor retinoid binding protein (IRBP), are also not fully understood. The role of IRBP in regulation of visual cycle may require receptors for transfer of retinoids. A long term goal is to identify such receptors for IRBP on the RPE and photoreceptor membrane surfaces. In the past year we have made progress in the following areas: 1) 4HPR induces apoptosis in ARPE-19 cells in a dose-and time-dependent manner through activation of caspases 2 and 3, and generation of reactive oxygen species (ROS). In addition, we found that the expression of heme oxygenase-1 (HO-1), a stress response protein, and the growth arrest and DNA damage-inducible transcription factor 153 (Gadd153) were increased in response to the ROS generation. Furthermore, RAR antagonists were able to block the 4HPR-induced ROS generation, the expression of its downstream mediator Gadd153, and apoptosis. These results clearly demonstrate that 4HPR induces apoptosis in human retinal pigment epithelial cells and that RARs mediate this process by regulating ROS generation as well as the expression of Gadd153 and HO-1. 2) 4HPR-induced neuronal differentiation of ARPE-19 cells is mediated through an MAPK/ERK1/2 signal transduction pathway. In addition, we have identified a number of genes that are differentially expressed in 4HPR-induced neuronal type differentiation of RPE cells as well as in apoptosis. IGFBP5, one such gene down regulated in 4HPR induced differentiation is known to regulate the signal transduction pathway mediated by IGF-1, which is involved in cell growth, differentiation and apoptosis. AGN194301, a RAR? antagonist, blocked both the down and up regulations of IGFBP5 and 6 indicating the involvement of retinoid signaling. Exogenous IGFBP5 was unable to block both the decrease in IGFBP5 expression as well as the neuronall differentiation indicating that IGFBP5 may not be a direct mediator of the neuronal differentiation. 3) Protein-protein interaction involving NORPEG protein was investigated using the yeast-two hybrid (Y2H) system. The N-terminal 334 amino acid residues when used as a bait was found to interact with only one protein, the kinesin family member 1A (KIF1A). However, bait containing the C-terminal 105 amino acid residues interacted with more than 40 different proteins. The Y2H results are being verified using tandem affinity purification and co-immunoprecipitatation techniques. A novel protein domain was identified by sequence analysis, defining a family of genes that are structurally related to NORPEG. This domain was used as one of the baits for yeast-two-hybrid experiments. Results from the Y2H experiments were compared with the mass spec analysis of proteins co-precipitated with NORPEG. Binding partners for NORPEG were found among cytoskeletal components, plasma membrane adherence junctions, and the nuclear splicesomal machinery, all of which are consistent with the varied subcellular localizations observed for NORPEG by confocal microscopy. Confocal immunofluorescence analysis of native bovine retinal pigment epithelium with an anti-NORPEG antibody showed staining of adherens junctions, specialized structures by which epithelial cells connect with each other. We developed a rabbit monoclonal antibody against the human NORPEG protein. The antibody preparation specifically reacts with the 110 kDa protein band corresponding to the NORPEG on western blot. This antibody also shows immunoreactivity towards murine and bovine orthologs of NORPEG and promises be a useful reagent to study the function of this protein. 4) In collaboration with Koutalos and coworkers we showed that the rate of all-trans retinol removal from photoreceptors is dependent on concentration of IRBP, confirming that IRBP is the physiologically relevant carrier for this process. This provides futher evidence for the existence of a receptor on photoreceptors for IRBP. In other work to further explore role of IRBP in retinoid transport to and from the retina, we hypothesized that multiple variants or isoforms of IRBP may also play different roles. Two forms of IRBP have been identified since its earliest characterization. We have purified both forms and submitted them to glycan chain analysis. We found that they have different chain composition.

RPE是位于光感受器和绒毛膜毛细血管之间的高度分化上皮细胞的单层，暴露于各种压力，包括暴露于光，炎症介质和活性氧。氧化应激增加导致的凋亡RPE细胞死亡可能会加速与年龄相关的黄斑变性（AMRD）的发作。源自维生素A的氧化的视黄酸会影响许多细胞功能，包括细胞生长，分化和凋亡。 这种作用是通过核激素受体RAR和RXR的转录调节介导的，维甲酸是配体。 视黄酸的合成类似物对细胞功能也有重大影响。 一种类似的类似物，fenretinide，N-（4-羟基苯基）视网膜化酰胺（4HPR），长期以来一直用作癌症预防剂。最近，已提出它是基于脂肪霉素的视网膜疾病的治疗剂。 在低剂量下，我们已经表明4HPR诱导培养的ARPE-19人类视网膜色素上皮细胞的神经元分化。 在较高剂量时会导致凋亡。 Norpeg（新型视网膜色素上皮细胞基因，RAI14），这是我们最初从人类视网膜色素上皮（RPE）细胞系ARPE-19表征的另一种视黄酸调节基因，它编码由980氨基酸残基组成的蛋白质。 Ankyrin重复区域和盘绕螺旋结构域的存在，即与蛋白质蛋白相互作用有关的结构结构域，这表明Norpeg与多个结合伴侣相关。 在这方面，这可能是重要的组织蛋白。 尽管大多数（如果不是全部），已经确定了视觉循环的酶促或结合蛋白成分，但视觉周期中的信号传导事件受到了较少的关注。 可以预料，视觉周期类维生素的通量受到诸如白天/夜间状态，环境光水平以及类维质异构体的相对水平等外部刺激的调节。 受体介导的全反式视黄醇的摄取以及11张视网膜的分泌，均可能涉及photecectopector veninoid结合蛋白（IRBP），也未完全了解。 IRBP在视觉周期调节中的作用可能需要受体转移类维生素类似。 一个长期的目标是在RPE和感光膜表面上识别IRBP的这种受体。 在过去的一年中，我们在以下领域取得了进展： 1）4HPR通过激活胱天蛋白酶2和3的激活，并产生活性氧（ROS），以剂量和时间依赖性的方式诱导ARPE-19细胞凋亡。 此外，我们发现血红素加氧酶-1（HO-1），应力反应蛋白以及生长停滞和DNA损伤诱导转录因子153（GADD153）的表达增加了ROS的产生。此外，RAR拮抗剂能够阻止4HPR诱导的ROS产生，其下游介体GADD153和凋亡。这些结果清楚地表明，4HPR诱导人视网膜色素上皮细胞的凋亡，并且通过调节ROS的产生以及GADD153和HO-1的表达来刺激这一过程。 2）ARPE-19细胞的4HPR诱导的神经元分化是通过MAPK/ERK1/2信号转导途径介导的。此外，我们已经确定了许多在4HPR诱导的RPE细胞和凋亡中差异表达的基因。 已知在4HPR诱导的分化中调节的基因IGFBP5可以调节由IGF-1介导的信号转导途径，IGF-1参与细胞生长，分化和凋亡。 AGN194301，RAR？拮抗剂，阻止了IGFBP5和6的向下和向上法规，表明视网膜类似信号的参与。 外源性IGFBP5无法阻止IGFBP5表达的降低以及神经元分化，这表明IGFBP5可能不是神经元分化的直接介体。 3）使用酵母 - 两种杂交（Y2H）系统研究了涉及NORPEG蛋白的蛋白质 - 蛋白质相互作用。 N末端334氨基酸残基被发现用作诱饵，仅与一种蛋白质，驱动蛋白家族成员1A（KIF1A）相互作用。但是，含有C末端105氨基酸残基的诱饵与40多种不同蛋白质相互作用。使用串联亲和力纯化和共免疫沉淀技术验证了Y2H的结果。 通过序列分析确定了一种新型的蛋白质结构域，定义了与NORPEG结构相关的基因家族。 该结构域被用作酵母 - 两个杂交实验的诱饵之一。 将Y2H实验的结果与与NORPEG共沉淀的蛋白质的质谱分析进行了比较。 在细胞骨架成分，质膜依从性连接和核显微镜机制中发现了NORPEG的结合伴侣，所有这些结合伴侣与共核显微镜观察到的不同亚细胞定位一致。对天然牛视网膜色素上皮的共聚焦免疫荧光分析与抗诺佩格抗体显示了粘附连接的染色，这些结构是上皮细胞相互连接的专门结构。我们开发了针对人Norpeg蛋白的兔单克隆抗体。抗体制备与与蛋白质印迹上的Norpeg相对应的110 kDa蛋白带特别反应。该抗体还显示出对Norpeg的鼠和牛直系同源物的免疫反应性，并承诺是研究该蛋白质功能的有用试剂。 4）与Koutalos及其同事合作，我们表明，从感光体中除去全反式视黄醇的速率取决于IRBP的浓度，证实IRBP是该过程中与生理上相关的载体。这为IRBP的感光体上存在受体提供了证据。 在进一步探索IRBP在视网膜类似往返视网膜中的角色中的作用的其他工作中，我们假设IRBP的多种变体或同工型也可能扮演不同的角色。 自从最早的表征以来，已经确定了两种形式的IRBP。 我们已经纯化了这两种形式，并将它们提交给聚糖链分析。 我们发现它们具有不同的链组成。