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: 7968375
• 负责人: Thomas Redmond
• 金额: $ 132.6万
• 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/7968375
• 关键词: 11 cis Retinal; Affect; Age related macular degeneration; All-Trans-Retinol; Apoptosis; Apoptotic; Appearance; Area; Asparagine; Attention; Be++ element; Beryllium; Binding Proteins; CCAAT-Enhancer-Binding Proteins; CCL2 gene; CXCL10 gene; CXCL9 gene; Cell Death; Cell physiology; Cells; Collaborations; Complex; Data; Degenerative Disorder; Development; Diabetic Retinopathy; Dose; Down-Regulation; Elements; Epithelial Cells; Event; Exposure to; Fenretinide; Gene Expression; Genes; Goals; Hand; Histocompatibility Testing; Homeostasis; Human; Human Activities; Hybrids; IGFBP5 gene; Immunity; Individual; Infection; Inflammation Mediators; Inflammatory; Insulin-Like Growth Factor I; Interleukin-6; Isomerism; Ligands; Light; Link; Lipofuscin; MAP Kinase Gene; Malignant Neoplasms; Mediating; Mediator of activation protein; Membrane; Metabolism; MicroRNAs; Microarray Analysis; Modification; Monosaccharides; Morphology; Neuronal Differentiation; Neurons; Nuclear Hormone Receptors; Oxidative Stress; Pathology; Pathway interactions; Phosphorylation; Phosphorylation Site; Photoreceptors; Play; Polysaccharides; Population; Preventive; Process; Protein Isoforms; Proteins; RANTES; RXR; Reactive Oxygen Species; Recombinants; Regulation; Regulator Genes; Relative (related person); Retina; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Retinoids; Role; Signal Transduction; Signal Transduction Pathway; Stimulus; Stress; Structure; Structure of retinal pigment epithelium; Surface; Therapeutic Agents; Time; Transcript; Transcriptional Regulation; Tretinoin; U-0126; Variant; Vitamin A; analog; base; cell growth; chemokine; cytokine; glycosylation; inhibitor/antagonist; interest; interstitial retinol-binding protein; monolayer; overexpression; oxidation; promoter; receptor; research study; response; retinamide; tumorigenesis; uptake; visual cycle

TThe 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 (AMD). 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. We are interested in how these effects of 4HPR are mediated. In another avenue of regulation, microRNAs (miRNAs) have received much attention as post-transcriptional regulators of gene expression in all cell/tissue types. The extent of their importance is just beginning to be realized. 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 ARPE19 cells due to agents with which they are treated in our experiments. 

Lastly, 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) We previously found that 4HPR-induced neuronal differentiation of ARPE-19 cells is mediated through an MAPK/ERK1/2 signal transduction pathway. In addition, we 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. In the past year we found that the neuronal differentiation of cultured human RPE cells induced by 4HPR is associated with down regulation of IGFBP5. This down regulation of IGFBP5 is inhibited by U0126, an inhibitor of MEK1/2, indicating the involvement of MAPK pathway. The ectopic addition of recombinant IGFBP5 is unable to block both the decrease in IGFBP5 expression as well as the neuronal differentiation, indicating that IGFBP5 may not be a direct mediator of the neuronal differentiation. On the other hand, overexpression of C/EBPβ (CCAAT/enhancer binding proteins) not only blocks the appearance of neuron-like morphology but also the observed decrease in the expression of IGFBP5. The activity of the human IGFBP5 promoter that contains the C/EBP element was decreased by 4HPR by approximately 2.5-fold as compared to basal conditions. Deleting C/EBP element from the IGFBP5 promoter abolished the activity in both basal and differentiating conditions. These data suggest that C/EBP regulates IGFBP5 expression at transcriptional level. 
 2) Inflammatory processes are known to be involved in the development of age-related macular degeneration (AMD). Human retinal pigment epithelial (HRPE) cells in cultures respond to inflammatory cytokines by increasing the expression of many chemokines and cytokines. Therefore, we investigated the role of miRNA in this process in collaboration with Chandra Nagineni and John Hooks (NEI-LI). By microarray analysis, miR-155 was identified as the predominant miRNA induced by cytokine mix (IFN-γ, TNF-α and IL-1β) in HRPE cells. Real-time PCR analysis showed that the miR-155 expression increased 10-fold under this condition. The observed alteration in miR-155 expression by the cytokine mix was associated with marked increases in the expression of CCL2, CCL5, CXCL9, CXCL10 and IL-6 transcripts as well as corresponding proteins. Individual treatment with IFN-γ, TNF-α or IL-1β showed noticeable but small increase in miR-155 expression, but combination of any two exaggerated this effect. These results show that inflammatory cytokines can modulate miR-155 expression in human RPE cells. Thus, miR-155, an miRNA implicated in immunity, infection and oncogenesis, could play an important role in inflammatory processes leading to AMD or other retinal degenerative diseases. 3) We have hypothesized that multiple variants or isoforms of IRBP may play different roles in the function of IRBP, and two forms of IRBP have been identified since its earliest characterization (IRBP I and II). It is possible that this is due to differential glycosylation of different populations and may have a functional relevance. In the past year we extended our studies of this modification. While no differences were noted in the monosaccharide composition of IRBP I and II, and IRBP I and II had similar asparagine-linked glycans, the proportion of each glycan differed between IRBP I and II. That is, the main asparagine-linked component of IRBP I was a complex type glycan, whereas, that of IRBP II was a hybrid type structure. The glycan structures of IRBP I and II may be important in the function of IRBP in the IPM. Another possible mechanism for IRBP post-translational modification is its phosphorylation. We are trying to identify which potential phosphorylation sites are utilized. The size of IRBP is making this task more difficult than anticipated. The relationship of phosphorylation to the differential glycosylation is also being investigated.

RPE是位于感光体和绒毛膜毛细血管之间高度分化的上皮细胞的单层，暴露于各种应激，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细胞死亡会加速与年龄相关的黄斑变性（AMD）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。我们对4HPR的这些效果如何介导感兴趣。 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。他们的重要性范围才刚刚开始实现。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，因此我们有兴趣确定ARPE19细胞中miRNA表达的变化，因为它们在我们的实验中对其进行了处理。 

最后，尽管已经确定了视觉周期的酶促或结合蛋白成分的大多数（如果不是全部），但视觉周期中的信号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）我们以前发现，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，我们确定了许多在4HPR诱导的RPE细胞神经元型分化以及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。在过去的一年中，我们发现由4HPR诱导的培养的人RPE细胞的神经元分化与IGFBP5的下调有关。 MEK1/2的抑制剂U0126抑制了IGFBP5的下降调节，表明MAPK途径的参与。重组IGFBP5的异位添加无法阻止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。另一方面，C/EBPβ（CCAAT/增强子结合蛋白）的过表达不仅阻止了神经元样形态的外观，而且还可以观察到IGFBP5表达的降低。与基础条件相比，包含C/EBP元件的人IGFBP5启动子的活性减少了约2.5倍。从IGFBP5启动子中删除C/EBP元素在基础和分化条件下都消除了活性。这些数据表明C/EBP在转录级别调节IGFBP5的表达。 
 2）已知炎症过程参与与年龄相关的黄斑变性（AMD）的发展。培养物中人类视网膜色素上皮细胞（HRPE）细胞通过增加许多趋化因子和细胞因子的表达来应对炎症性细胞因子。因此，我们研究了miRNA在与Chandra Nagineni和John Hooks（Nei-Li）合作中的作用。通过微阵列分析，将miR-155鉴定为HRPE细胞中细胞因子混合物（IFN-γ，TNF-α和IL-1β）诱导的主要miRNA。实时PCR分析表明，在这种情况下，miR-155表达增加了10倍。观察到的通过细胞因子混合物在miR-155表达中的改变与CCL2，CCL5，CXCL9，CXCL10和IL-6转录本的表达显着增加以及相应的蛋白质相关。用IFN-γ，TNF-α或IL-1β进行的单个治疗表现出明显的，但miR-155表达的增加，但两种组合的组合夸大了这种效果。这些结果表明，炎性细胞因子可以调节人RPE细胞中的miR-155表达。因此，与免疫，感染和肿瘤发生有关的miRNA可能在导致AMD或其他视网膜退行性疾病的炎症过程中起重要作用。 3）我们假设IRBP的多种变体或同工型可能在IRBP的功能中起不同的作用，并且自从最早的表征（IRBP I和II）以来已经确定了两种形式的IRBP。这可能是由于不同人群的差异糖基化引起的，并且可能具有功能相关性。在过去的一年中，我们扩展了对这种修改的研究。 尽管IRBP I和II的单糖组成中没有发现差异，而IRBP I和II具有相似的天冬酰胺连接聚糖，但IRBP I和II之间每个聚糖的比例都不同。也就是说，IRBP I的主要天冬酰胺连接的组件是一种复杂的聚糖，而IRBP II的组成部分是一种杂交类型的结构。 IRBP I和II的聚糖结构在IPM中IRBP的功能中可能很重要。 IRBP翻译后修饰的另一种可能的机制是其磷酸化。我们正在尝试确定利用哪些潜在的磷酸化位点。 IRBP的规模使这项任务比预期的要困难。 还研究了磷酸化与差糖基化的关系。