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: 8938324
• 负责人: Thomas Redmond
• 金额: $ 109.1万
• 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/8938324
• 关键词: Adult; Affect; Age related macular degeneration; Anti-Inflammatory Agents; Apoptosis; Apoptotic; Area; Attention; Binding Proteins; Biochemistry; Bioinformatics; Biology; Blocking Antibodies; Cattle; Cell Culture Techniques; Cell Death; Cell Line; Cell Therapy; Cell physiology; Cells; Cellular Morphology; Cellular biology; Competence; Disease; Down-Regulation; Employee Strikes; Ensure; Enzymes; Epithelial; Event; Extramural Activities; Eye; Eye diseases; Fenretinide; Functional disorder; GRB10 gene; GRP78 gene; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Genes; Genetic; Goals; High Pressure Liquid Chromatography; Human; Immunoprecipitation; Inflammation; Inflammatory; Inflammatory Response; Interferons; Interleukin-1; Isomerism; Laboratories; Light; Lipids; Lipofuscin; MG132; Manuscripts; Mass Spectrum Analysis; Mediating; Mesenchyme; Metabolism; Methods; MicroRNAs; Modeling; Modification; Molecular Biology; Molecular Structure; Monounsaturated Fatty Acids; Non-Insulin-Dependent Diabetes Mellitus; Oxidative Stress; Pathogenesis; Pathway interactions; Pattern; Phenotype; Phosphopeptides; Phosphorylation; Physiology; Play; Process; Proteasome Inhibitor; Proteins; Publishing; RNA; Regulation; Regulator Genes; Relative (related person); Reporting; Research; Resveratrol; Retina; Retinal Degeneration; Retinal Diseases; Retinoids; Role; Signal Pathway; Signal Transduction; Stearoyl-CoA Desaturase; Stimulus; Stress; Structure of retinal pigment epithelium; Thapsigargin; Therapeutic; Time; Tissues; Transcript; Transplantation; Tretinoin; Tumor Necrosis Factor-alpha; Tunicamycin; Ubiquitin; Ubiquitin-Activating Enzymes; Ubiquitination; Work; analog; base; cancer cell; cancer type; cell growth; cytokine; disorder of macula of retina; fetal; functional genomics; improved; in vivo; induced pluripotent stem cell; inhibitor/antagonist; insulin sensitivity; interest; monolayer; multicatalytic endopeptidase complex; research study; response; retinamide; small molecule; stem; visual cycle

We are studying signaling networks in the retinal pigment epithelium (RPE) with special emphasis on lipid and retinoid metabolism pathways, differentiation/dedifferentiation pathways, and protection against oxidative stress. Apoptotic RPE cell death resulting from increased oxidative stress could hasten the onset of age-related macular degeneration (AMD) and may be regulated by retinoic acid (RA). RA affects many cellular functions including cell growth, differentiation, and apoptosis. Synthetic analogs of retinoic acid also have significant effects on cellular function and are being studied. Also, the role of signaling pathways in RPE differentiation and de-differentiation is an important focus of our research. Divergence from or convergence to the phenotype of native RPE is a common theme of much RPE cell culture research and this has an important impact on the potential use of RPE cells in cell therapy for retinal degenerations. In addition, given the likely importance of microRNAs (miRNAs) as post-transcriptional regulators of gene expression in the response of RPE cells to various signals, we are interested in determining changes in miRNA expression in RPE cells due to agents with which they are treated in our experiments. In the past year we have made progress in the following areas: 1) Stearoyl-CoA desaturase (SCD) regulates cellular functions by controlling the ratio of saturated to monounsaturated fatty acids. Increase in SCD expression is strongly implicated in the proliferation and survival of cancer cells, whereas its decrease is known to impair proliferation, induce apoptosis, and restore insulin sensitivity. We examined whether fenretinide, which induces apoptosis in cancer cells and recently shown to improve insulin sensitivity, can modulate the expression of SCD. We observed that fenretinide decreased SCD protein and enzymatic activity in ARPE-19 cells. Increased expression of BiP/GRP78, ATF4 and GADD153 implicated ER stress. Tunicamycin and thapsigargin, compounds known to induce ER stress, also decreased the SCD protein. This decrease was completely blocked by the proteasome inhibitor MG132. In addition, PYR-41, an inhibitor of ubiquitin activating enzyme E1, blocked the fenretinide-mediated decrease in SCD. Immunoprecipitation analysis using anti-ubiquitin and anti-SCD antibodies and the blocking of SCD loss by PYR41 inhibition of ubiquitination further corroborate that fenretinide mediates the degradation of SCD in human RPE cells via the ubiquitin-proteasome dependent pathway. Therefore, the effect of fenretinide on SCD should be considered in its potential therapeutic role against cancer, type-2 diabetes, and retinal diseases such as AMD. A manuscript describing this work was published in this reporting period. 2) We continued our work on the role of miRNAs in regulating the inflammatory response of the retinal pigment epithelium (RPE), implicated in the pathogenesis of age-related macular degeneration. The role of microRNAs miR-146a and miR-146b-5p in regulation of the inflammatory process induced by the proinflammatory cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β in human RPE cells has been studied. We are also conducting experiments to modulate the effect of proinflammatory cytokines on the inflammatory response using anti-inflammatory agents such as resveratrol. These experiments have been extended to long-term mature confluent ARPE-19 monolayers to evaluate the effect of proinflammatory cytokines on miRNA expression in differentiated RPE cells. 3) We have expanded a study to understand the mechanisms underlying dedifferentiation of RPE cells in primary culture and redifferentiation in the ARPE-19 RPE cell line. Divergence from or convergence to the phenotype of native RPE is a common theme of much RPE cell culture research. On the one hand, using a cocktail of factors induced pluripotent stem (iPS) cells can be differentiated into cells sharing many aspects of RPE phenotype, and by rigorous culture methods, fetal RPE cells can be differentiated to retain or acquire aspects of native phenotype. On the other hand, explanted native RPE cells will lose important aspects of their RPE phenotype after a short time in culture. The various cell lines, such as the commonly used ARPE-19, do not have most native phenotypic features under common culture methods. What are the mechanisms regulating such gain or loss? Do mechanisms like epithelial-mesenchyme transition play a role in this process? We are particularly interested in the long-known but poorly understood loss by immortalized and primary RPE cells of expression of visual cycle enzymes. Understanding the mechanism underlying this down-regulation could be useful in ensuring that iPS-derived cells used for human transplant are fully competent to fulfill their intended role in restoring RPE function in treated eyes. Our experimental paradigm focuses i) on the loss of visual cycle phenotypic competence by adult bovine RPE cells explanted into primary culture, and ii) the regain of phenotypic competence by careful culture of relatively early passage ARPE-19 cells. Using these models we are analyzing expression of visual cycle and genes of other pathways and will correlate these to changes in gene regulation, RNA transcript expression and microRNA expression patterns. Striking changes in phenotype (cell morphology and melanization), gene expression, and biochemistry have been observed in ARPE-19 cells grown for 4 months or more, and indicate that ARPE-19 retains the plasticity to return to native-like RPE phenotype. The study is still ongoing. 4) We continued analysis of post-transcriptional modifications of IRBP. In this reporting period we have continued to study whether phosphorylation of IRBP occurs in vivo. An HPLC method to purify phosphopeptides has been established and these are being analyzed by mass spectrometry. We continue to collaborate within the LRCMB and with other laboratories and sections (LI, Molecular Structure and Functional Genomics), as well as with extramural labs in the analysis of retinoid and other compounds. A manuscript on the latter was published during this reporting period.

我们正在研究视网膜色素上皮（RPE）中的信号网络，并特别强调了脂质和维生素代谢途径，分化/去分化途径以及防止氧化应激的保护。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（AMD）的发作，并且可以通过视黄酸（RA）调节。 RA会影响许多细胞功能，包括细胞生长，分化和凋亡。视黄酸的合成类似物对细胞功能也有显着影响，并正在研究。同样，信号通路在RPE分化和脱不同的作用中的作用是我们研究的重要重点。从或收敛到天然RPE的表型的差异是大量RPE细胞培养研究的一个共同主题，这对RPE细胞在细胞疗法中的潜在用途具有重要影响。此外，鉴于MicroRNA（miRNA）可能是RPE细胞对各种信号反应中基因表达的转录后调节剂的重要性，因此由于在我们的实验中对其进行了治疗，我们有兴趣确定RPE细胞中miRNA表达的变化。 在过去的一年中，我们在以下领域取得了进展： 1）stearoyl-COA去饱和酶（SCD）通过控制饱和单不饱和脂肪酸的比率来调节细胞功能。 SCD表达的增加与癌细胞的增殖和存活有很大的影响，而其减少却损害了增殖，诱导凋亡和恢复胰岛素敏感性。我们检查了诱导癌细胞凋亡并最近证明可以提高胰岛素敏感性的芬肠丁胺是否可以调节SCD的表达。我们观察到fenretinide在ARPE-19细胞中降低了SCD蛋白和酶促活性。 BIP/GRP78，ATF4和GADD153的表达增加暗示了ER应力。皮其中霉素和Thapsigargin，已知可诱导ER应激的化合物也降低了SCD蛋白。蛋白酶体抑制剂MG132完全阻止了这种减少。此外，泛素激活酶E1的抑制剂PyR-41阻断了fenretinide介导的SCD降低。使用抗泛素和抗SCD抗体的免疫沉淀分析，以及通过PYR41抑制泛素化对SCD损失的阻断，进一步证实了Fenretinide通过泛素 - 蛋白 - 蛋白质组依赖性途径介导人RPE细胞中SCD降解的降解。因此，应考虑其对癌症，2型糖尿病和视网膜疾病（如AMD）的潜在治疗作用，应考虑fenretinide对SCD的影响。 描述这项工作的手稿在此报告期间发表。 2）我们继续进行有关miRNA在调节视网膜色素上皮（RPE）炎症反应中的作用的工作，这与年龄相关的黄斑变性的发病机理有关。已经研究了人RPE细胞中microRNAS miR-146a和miR-146b-5p在调节由促炎细胞因子干扰素（IFN）-γ，肿瘤坏死因子（TNF）-α和interleukin（IL）-1β引起的炎症过程中的作用。 我们还正在进行实验，以调节使用抗炎药（如白藜芦醇）调节促炎细胞因子对炎症反应的影响。这些实验已扩展到长期成熟的汇合ARPE-19单层，以评估促炎细胞因子对分化RPE细胞中miRNA表达的影响。 3）我们已经扩展了一项研究，以了解RPE细胞在原代培养物中的去分化的基础机制，并在ARPE-19 RPE细胞系中重新分化。 从或收敛到天然RPE表型的差异是许多RPE细胞培养研究的共同主题。 一方面，使用一系列因子诱导多能茎（IPS）细胞可以区分共享RPE表型的许多方面的细胞，并且通过严格的培养方法，可以将胎儿RPE细胞区分开以保留或获取天然表型的方面。另一方面，经过短暂的培养时间后，外植的天然RPE细胞将失去其RPE表型的重要方面。各种细胞系（例如常用的ARPE-19）在共同培养方法下没有大多数天然表型特征。 调节这种收益或损失的机制是什么？ 上皮 - 间质转变等机制是否在此过程中起作用？ 我们对视觉循环酶表达的永生和原代RPE细胞的众所周知但知之甚少的损失特别感兴趣。 了解这种下调的基础机制对于确保用于人类移植的IPS衍生细胞具有完全有能力履行其在恢复治疗眼中RPE功能中的预期作用的机制。 我们的实验范式重点是i）成年牛RPE细胞的视觉周期表型能力的丧失，而ii）ii）通过仔细的早期通过ARPE-19细胞的仔细培养来恢复表型能力。使用这些模型，我们正在分析其他途径的视觉循环和基因的表达，并将其与基因调节，RNA转录表达和microRNA表达模式的变化相关。已经在ARPE-19细胞中观察到了表型（细胞形态和黑色素化），基因表达和生物化学的惊人变化，在ARPE-19细胞中生长了4个月或更长时间，并表明ARPE-19保留可塑性，以返回到本机样RPE表型。该研究仍在进行中。 4）我们继续分析IRBP的转录后修饰。 在此报告期间，我们继续研究IRBP的磷酸化是否发生在体内。 已经建立了一种纯化磷酸肽的HPLC方法，并通过质谱法分析了这些方法。我们继续在LRCMB内以及其他实验室和切片（LI，分子结构和功能基因组学），以及在分析性类维生素类和其他化合物的分析中与外壁外实验室合作。在此报告期间，在后者上发表了一份手稿。