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: 8556839
• 负责人: Thomas Redmond
• 金额: $ 109.58万
• 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/8556839
• 关键词: 11 cis Retinal; Adult; Affect; Age related macular degeneration; All-Trans-Retinol; Antibodies; Apoptosis; Apoptotic; Area; Attention; Binding Proteins; Biochemical; Biochemistry; Bioinformatics; Biological Assay; CCAAT-Enhancer-Binding Proteins; CCL2 gene; CXCL10 gene; CXCL9 gene; Cattle; Cell Culture Techniques; Cell Death; Cell Line; Cell physiology; Cells; Cellular biology; Ceramides; Competence; Cytokine Signaling; Data; Degenerative Disorder; Disease; Down-Regulation; Endoplasmic Reticulum; Ensure; Enzymes; Epithelial; Eukaryotic Cell; Event; Eye; Eye diseases; Fatty Acids; Fenretinide; Functional disorder; Gene Expression; Gene Expression Regulation; Genes; Genetic; Goals; High Pressure Liquid Chromatography; Histocompatibility Testing; Human; IRAK1 gene; Immunoprecipitation; Inflammation; Inflammatory; Inflammatory Response; Insulin-Like Growth Factor Binding Protein 5; Interferons; Interleukin-1; Interleukin-6; Isomerism; Laboratories; Lead; Light; Lipids; Lipofuscin; MAP Kinase Gene; MG132; Malignant Neoplasms; Manuscripts; Mass Spectrum Analysis; Mediating; Membrane; Mesenchyme; Metabolism; Methods; MicroRNAs; Modeling; Modification; Molecular Biology; Molecular Structure; Neuronal Differentiation; Organelles; Oxidative Stress; Pathogenesis; Pathway interactions; Pattern; Phenotype; Photoreceptors; Physiological; Play; Preparation; Preventive; Process; Production; Proteasome Inhibitor; Proteins; RANTES; RNA; Recombinants; Regulation; Regulator Genes; Relative (related person); Research; Retina; Retinal Degeneration; Retinal Diseases; Retinoids; Role; Signal Pathway; Signal Transduction; Sphingolipids; Stearoyl-CoA Desaturase; Stimulus; Structure of retinal pigment epithelium; Surface; TNF gene; Thapsigargin; Therapeutic Agents; Time; Transcript; Translational Repression; Transplantation; Tretinoin; Tunicamycin; Ubiquitin; Ubiquitin-Activating Enzymes; Ubiquitination; Unsaturated Fatty Acids; Work; analog; base; cell growth; cytokine; disorder of macula of retina; endoplasmic reticulum stress; fetal; functional genomics; immortalized cell; induced pluripotent stem cell; inhibitor/antagonist; interest; interstitial retinol-binding protein; multicatalytic endopeptidase complex; overexpression; promoter; protein degradation; protein expression; protein folding; receptor; research study; response; retinamide; small molecule; stem; uptake; visual cycle

We wish to develop an understanding of signaling networks in the retinal pigment epithelium (RPE) with special emphasis on retinoid metabolism pathways and protection against light damage and 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. One such analog, fenretinide (N-(4-hydoxyphenyl)retinamide; 4HPR), has been used as a cancer preventive agent and has been proposed as a therapeutic agent for lipofuscin-based retinal diseases, and we are interested in how these effects of 4HPR are mediated. We previously found that neuronal differentiation of ARPE-19 cells induced by 4HPR was mediated by the MAPK pathway and that this was associated with decreased expression of insulin-like growth factor binding protein-5 under the regulation of CCAAT/enhancer-binding protein. Stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the synthesis of unsaturated fatty acids, is also involved in 4HPR-induced effects on RPE cells. SCD plays an important early role in the synthesis of sphingolipids and ceramides, important effectors in cellular biology. A close association between the production of ceramide and the onset of programmed cell death has been well established. We are interested in the role of SCD in RPE biochemistry. In another avenue of regulation, microRNAs (miRNAs) have received much attention as post-transcriptional regulators of gene expression in all cell/tissue types. Given the likely importance of this level of regulation 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. We previously found that 4HPR increased expression of microRNA-9, and that inflammatory cytokines regulate microRNA-155 expression in human retinal pigment epithelial cells by activating the JAK/STAT pathway. In the past year we have made progress in the following areas: 1) We continued to study the role of SCD in 4HPR-induced apoptosis of RPE cells. Endoplasmic reticulum (ER) is the central organelle in eukaryotic cell for lipid synthesis, protein folding and maturation. A number of biochemical and physiological stimuli induces ER stress, and if unchecked could lead to apoptosis. Recombinant SCD protein is degraded via the ubiquitin-proteasome-dependent pathway when overexpressed in cells. It is also known that ER stress induces ubiquitin-proteasome-dependent degradation of proteins. We examined whether ER stress affects the SCD expression. Human ARPE-19 cells treated with 4HPR, tunicamycin or thapsigargin, compounds that induce ER stress, showed a time-dependent decrease in the expression of SCD protein, while markers of ER stress were markedly increased. The decrease in SCD protein expression was completely blocked by the proteasome inhibitor, MG132. In addition, PYR-41, an irreversible inhibitor of ubiquitin activating enzyme E1, also completely blocked the 4HPR-induced decrease in SCD protein expression. Immunoprecipitation analysis of 4HPR-treated cell lysate using either ubiquitin or SCD antibody showed that ER stress increased the ubiquitination of proteins including SCD. These data show that ER stress mediates the degradation of SCD in human RPE cells via the ubiquitin-proteasome dependent pathway. A manuscript describing these data is currently in preparation. 2) We have continued our work on the role of miRNAs in regulating the inflammatory response of adult human RPE cells. Inflammatory response of the RPE is implicated in the pathogenesis of age-related macular degeneration (AMD). The microRNAs miR-146a and miR-146b-5p are known to control the inflammatory process by their ability to regulate key genes involved in cytokine signaling by translational repression. We have investigated the expression of miR-146a and miR-146b-5p in human RPE cells and their response to pro-inflammatory cytokines. Real-time PCR analysis of confluent cultures of RPE cells established from adult human donor eyes showed that miR-146a and 146b-5p are expressed in RPE cells. The cells responded to pro-inflammatory cytokines (IFN-γ + TNF-α + IL-1β) by highly increasing the expression of both miR-146a and miR-146b-5p. This was associated with an increase in the expression of transcripts for CCL2, CCL5, CXCL9, CXCL10 and IL-6, and a decrease in that for HMOX1. The miR-146a induction was dependent on IL-1β since its omission from the cytokine mix resulted in a greatly reduced response. In contrast, the induction of miR-146b-5p was dependent on IFN-γ since its omission from the cytokine mix minimized the effect. Also, the increase in MIR146B promoter activity by the cytokine mix was effectively blocked by JAK inhibitor 1, a known inhibitor of JAK/STAT signaling pathway. The expression of IRAK1 protein was decreased when ARPE-19 cells were transiently transfected with miR-146a or miR-146b-5p mimics. These results show that both miR-146a and miR-146b-5p are expressed in human RPE cells in culture and their expression is regulated by pro-inflammatory cytokines, miR-146a being dependent on IL-1β and miR-146b-5p on IFN-γ. These two microRNAs could play a role in inflammatory processes underlying AMD or other retinal degenerative diseases by their ability to regulate IRAK1 expression. A manuscript describing these data is currently in preparation. 3) We continued a study to understand the mechanisms underlying dedifferentiation of RPE cells in primary culture. Divergence from or convergence to the phenotype of native RPE is a common theme of much RPE cell culture research. On the one hand, 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 immortalized cell lines, such as the commonly used ARPE-19, have lost most native phenotypic features. 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 on the loss of visual cycle competence by adult bovine RPE cells explanted into primary culture. We have developed methods for establishing bovine RPE cells in primary culture. Using these we are analyzing expression of visual cycle and other genes and will correlate these to changes in gene regulation, RNA transcript expression and microRNA expression patterns. The study is still ongoing. 4) We have continued analysis of post-transcriptional modifications of IRBP. We have established non-radioactive HPLC/mass spectrometric assays for several fatty acids, sphingolipids and ceramides to assist in current research. In addition, we have collaborated within the LRCMB and with other laboratories and sections (LI, Molecular Structure and Functional Genomics) to provide retinoid and other analyses.

我们希望在视网膜色素上皮（RPE）中发展对信号网络的理解，并特别强调视网膜类似的代谢途径，并保护免受轻损伤和氧化应激。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）已被用作癌症预防剂，并已被提议用作基于脂肪霉素的视网膜疾病的治疗剂，我们对如何介导4HPR的这些影响感兴趣。我们先前发现，由MAPK途径介导了由4HPR诱导的ARPE-19细胞的神经元分化，这与CCAAT/增强子结合蛋白的调节下胰岛素样生长因子结合蛋白5的表达降低有关。 stearoyl-COA去饱和酶（SCD）是一种不饱和脂肪酸合成的限速酶，也参与了4HPR诱导的对RPE细胞的影响。 SCD在鞘脂和神经酰胺的合成中起着重要的早期作用，细胞生物学中的重要效应子。 神经酰胺的产生与程序性细胞死亡的发作之间的密切关联已经建立。 我们对SCD在RPE生物化学中的作用感兴趣。 在另一个调节途径中，MicroRNA（miRNA）在所有细胞/组织类型中都引起了基因表达的转录后调节剂的关注。鉴于这种调节水平在RPE细胞对各种信号的响应中的可能性可能很重要，因此由于在我们的实验中对其进行了处理，我们有兴趣确定RPE细胞中miRNA表达的变化。 我们先前发现4HPR增加了microRNA-9的表达，并且炎性细胞因子通过激活JAK/STAT途径来调节人视网膜色素上皮细胞中microRNA-155的表达。 在过去的一年中，我们在以下领域取得了进展： 1）我们继续研究SCD在4HPR诱导的RPE细胞凋亡中的作用。内质网（ER）是用于脂质合成，蛋白质折叠和成熟的真核细胞中的中央细胞器。许多生化和生理刺激会引起ER应激，如果未检查的话可能导致细胞凋亡。 重组SCD蛋白在细胞中过表达时通过泛素 - 蛋白酶体依赖性途径降解。还众所周知，ER应激会诱导蛋白质的泛素 - 蛋白酶体依赖性降解。 我们检查了ER应力是否影响SCD表达。用4HPR，衣霉素或Thapsigargin处理的人ARPE-19细胞诱导ER应激的化合物显示SCD蛋白表达的时间依赖性降低，而ER应力的标记显着增加。蛋白酶体抑制剂MG132完全阻断了SCD蛋白表达的降低。 此外，Pyr-41是一种不可逆的泛素激活酶E1的抑制剂，也完全阻断了4HPR诱导的SCD蛋白表达降低。使用泛素或SCD抗体对4HPR处理的细胞裂解物进行免疫沉淀分析表明，ER应激会增加蛋白质（包括SCD）的泛素化。这些数据表明，ER应力通过泛素 - 蛋白酶体依赖性途径介导了人RPE细胞中SCD的降解。 描述这些数据的手稿目前正在准备中。 2）我们继续在调节成年人类RPE细胞的炎症反应中的作用方面继续研究。 RPE的炎症反应与年龄相关的黄斑变性（AMD）的发病机理有关。已知MicroRNAS miR-146a和miR-146b-5p通过调节通过转化抑制参与细胞因子信号传导的关键基因的能力来控制炎症过程。我们已经研究了人RPE细胞中miR-146a和miR-146b-5p的表达及其对促炎性细胞因子的反应。实时PCR分析从成年人类供体眼中建立的RPE细胞的汇合培养物表明miR-146a和146b-5p在RPE细胞中表达。细胞对促炎性细胞因子（IFN-γ + TNF-α + IL-1β）做出了反应，通过高度增加miR-146a和miR-146b-5p的表达。这与CCL2，CCL5，CXCL9，CXCL10和IL-6的转录本表达的增加有关，而HMOX1的转录物表达也会增加。 miR-146a诱导取决于IL-1β，因为其细胞因子混合物的遗漏导致反应大大降低。相反，miR-146b-5p的诱导取决于IFN-γ，因为它从细胞因子混合物中遗漏了其最小化的效果。同样，JAK抑制剂1（一种已知的JAK/STAT信号途径抑制剂）有效阻断了细胞因子混合物的miR146b启动子活性的增加。当用miR-146a或miR-146b-5p模拟物瞬时转染ARPE-19细胞时，IRAK1蛋白的表达降低。 这些结果表明，miR-146a和miR-146b-5p均在培养物中的人RPE细胞中表达，其表达受促炎性细胞因子的调节，miR-146a依赖于IL-1β和IFN-γ的miR-146b-5p。这两种microRNA可以通过调节IRAK1表达的能力在AMD或其他视网膜退行性疾病的炎症过程中发挥作用。描述这些数据的手稿目前正在准备中。 3）我们继续进行一项研究，以了解原代培养物中RPE细胞的延分分化的机制。 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，诱导的多能茎（IPS）细胞可以分化为共享RPE表型许多方面的细胞，并且通过严格的培养方法，可以区分胎儿RPE细胞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。各种永生的细胞系，例如常用的ARPE-19，已经失去了最天然的表型特征。 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。 我们的实验范式着重于成年牛RPE细胞的视觉周期能力的丧失，这些细胞散布在原发性培养物中。 我们开发了在原发性培养中建立牛RPE细胞的方法。使用这些，我们正在分析视觉循环和其他基因的表达，并将其与基因调节，RNA转录表达和microRNA表达模式的变化相关。该研究仍在进行中。 4）我们继续对IRBP的转录后修饰进行分析。 我们已经建立了几种脂肪酸，鞘脂和神经酰胺的非放射性HPLC/质谱测定法，以协助当前研究。 此外，我们在LRCMB中以及其他实验室和切片（LI，分子结构和功能基因组学）合作，提供类类类线和其他分析。