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: 8149179
• 负责人: Thomas Redmond
• 金额: $ 135.17万
• 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/8149179
• 关键词: Adult; Affect; Age related macular degeneration; Apoptosis; Apoptotic; Area; Binding; Binding Proteins; Biochemical; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Proteins; Collaborations; Conjunctival Pterygium; Cornea; Data; Degenerative Disorder; Diabetic Retinopathy; Dose; Down-Regulation; Doxycycline; Elements; Enzymes; Epithelial Cells; Exposure to; Eye; Gel; Gene Expression; Genes; Goals; Growth; Histocompatibility Testing; Homeostasis; Human; IGFBP5 gene; Immigration; Indium; Inflammation Mediators; Inflammatory; Inflammatory Response; Interferon Type II; Isomerism; Keratin; Lesion; Ligands; Light; Lipids; Lipofuscin; MAP Kinase Gene; Metabolism; MicroRNAs; Microarray Analysis; Monounsaturated Fatty Acids; Mus; Neuronal Differentiation; Nuclear Extract; Oxidative Stress; Paper; Pathology; Pathway interactions; Photoreceptors; Play; Polyamines; Preventive; Process; Protein Binding; Publishing; RXR; Reactive Oxygen Species; Regulator Genes; Reporter; Retina; S100A9 gene; STAT1 gene; Signal Transduction; Stimulus; Structure of retinal pigment epithelium; Sun Exposure; Surface; Therapeutic Agents; Transcriptional Regulation; Tumor Necrosis Factor-alpha; Unsaturated Fatty Acids; analog; aquaporin 3; base; chromatin immunoprecipitation; enzyme activity; human TNF protein; inhibitor/antagonist; interstitial retinol-binding protein; keratin 12; protein expression; uptake

In the past year we have made progress in the following areas: 1) Efforts to identify the down regulation of IGFBP5 expression in 4-HPR-induced neuronal differentiation of human RPE (ARPE-19) cells continued and we showed that the decrease in IGFBP5 expression appears to be due to neuronal differentiation and is not a prerequisite for the neuronal differentiation of human RPE cells (Samuel et al., J. Cell Physiol. 224:827-836, 2010). We found that IGFBP5 down regulation is inhibited by U0126, an inhibitor of MEK1/2, indicating the involvement of MAPK pathway. The overexpression of transcription factor C/EBP (CCAAT/enhancer binding proteins) inhibited the 4-HPR-induced down regulation of IGFBP5 expression and the neuronal differentiation of RPE cells. Interestingly, the binding of C/EBPbeta to the IGFBP5 promoter was decreased by the 4-HPR treatment in gel shift and chromatin immunoprecipitation analyses. Further, the deletion of C/EBP response element from human IGFBP5 promoter markedly decreased the basal promoter activity and abolished its responsiveness to 4HPR treatment in reporter assays suggesting that the expression of IGFBP5 is regulated by C/EBP. We also continued efforts to identify the role of stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the synthesis of unsaturated fatty acids, in RPE cell death. SCD by its ability to regulate the ratio of saturated fatty acids to monounsaturated fatty acids is thought to control the structural integrity and fluidity of cell membranes, and thereby plays an important role in cell growth, differentiation and apoptosis. We observed that SCD protein expression and enzyme activity is decreased in human RPE cells during 4-HPR-induced apoptosis. We are investigating inhibition of 4HPR-induced apoptosis in human RPE cells by inhibitors of SCD by biochemical assays and microarray analysis of cultured RPE cells. We have modified and established HPLC and mass spectrometric assays for several lipid metabolites implicated in the SCD pathway and its role in cell growth, differentiation and apoptosis. 2) We have continued our work on the role of miRNA in regulating the inflammatory response of adult human retinal pigment epithelial (HRPE) cells in collaboration with Chandra Nagineni and John Hooks (NEI-LI). Our earlier work showed that the inflammatory cytokines (IFN-gamma + TNF-alpha + IL-1beta) can markedly increase the expression of miR-155 expression in HRPE cells. Although IFN-gamma is known to regulate gene expression by activating JAK/STAT signaling pathway, the role of this pathway in regulating the expression of miR-155 is not yet elucidated. Therefore, we studied the role of JAK/STAT pathway in mediating the regulation of miR-155 expression in HRPE cells by the inflammatory cytokines. We detected two putative STAT1 binding elements in the proximal region of BIC/miR-155 promoter. Electrophoretic mobility shift assays showed that increased protein binding to both these elements was present in nuclear extracts prepared from treated cells compared to control cells. The increase in miR-155 expression by the inflammatory cytokines was also associated with an increase in STAT1 activation. The increases in STAT1 activation, protein binding to STAT1 elements and miR-155 expression were effectively blocked by JAK inhibitor 1, an inhibitor of the JAK/STAT pathway. Thus, our results clearly show that the inflammatory cytokines increase miR-155 expression in human retinal pigment epithelial cells by directly activating the JAK/STAT signaling pathway. Thus, the modulation of miR-155 expression by the JAK/STAT signaling pathway could play an important role in the inflammatory processes leading to AMD or other retinal degenerative diseases. 3) The conclusion of a multi-lab collaborative investigation characterizing the processes associated with pterygium was reflected in 2 papers published in the past year. Pterygium is a vision-impairing fibrovascular lesion that grows across the corneal surface and is associated with sunlight exposure. We used expressed sequence tag analysis of an unnormalized unamplified pterygium cDNA library to examine the transcriptional repertoire of isolated pterygium and to identify marker genes for tissue origin and cell migration. Expression of selected genes was verified by immunofluorescence localization in normal eye ocular surface and in pterygium. The most abundant complementary DNAs from pterygium include clusterin, keratins 13 (Krt13) and 4 (Krt4), S100A9/calgranulin B, and spermidine/spermine N1-acetyltransferase (SAT1). Markers for both conjunctiva (such as keratin 13/4 and AQP3) and corneal epithelium (such as keratin 12/3 and AQP5) were present. Several of the genes expressed most abundantly in excised pterygium, particularly S100A9 and SAT1, have roles in cell migration. SAT1 exerts its effects through control of polyamine levels. IPENSpm, a polyamine analogue, showed a significant ability to reduce migration in primary cultures of pterygium. The expression pattern of keratins and other markers in pterygium most closely resemble those of conjunctival and limbal cells; some corneal markers are present, notably Krt12, but at lower levels than equivalent conjunctival markers. Our data are consistent with the model of pterygium developing from the migration of conjunctival- and limbal-like cells into corneal epithelium. Identification of genes with roles in cell migration suggests potential therapeutic targets. In particular, the ability of polyamine analogues to reduce migration in primary cultures of pterygium presents a possible approach to slowing pterygium growth. We also found that doxycycline causes regression of murine models of pterygium.

在过去的一年中，我们在以下领域取得了进展： 1）在4-HPR诱导的人RPE神经元分化（ARPE-19）细胞中IGFBP5表达的下降调节的努力继续进行，我们表明，IGFBP5表达的下降似乎是由于神经元分化的原因，并且似乎不是由于人类RPE细胞的神经元分化的前提（Samuel et andiol and and and and and and and and and。我们发现，MEK1/2的抑制剂U0126抑制IGFBP5降低调节，表明MAPK途径的参与。转录因子C/EBP（CCAAT/增强子结合蛋白）的过表达抑制了4-HPR诱导的下降调节IGFBP5表达和RPE细胞的神经元分化。有趣的是，在凝胶移位和染色质免疫沉淀分析中，4-HPR处理降低了C/EBPBETA与IGFBP5启动子的结合。此外，人IGFBP5启动子的C/EBP响应元件的缺失显着降低了基础启动子的活性，并在记者测定中消除了其对4HPR治疗的反应性，这表明IGFBP5的表达受C/EBP调节。我们还继续努力确定stearoyl-COA去饱和酶（SCD）的作用，这是RPE细胞死亡中不饱和脂肪酸合成中限速酶的作用。 SCD通过调节饱和脂肪酸与单不饱和脂肪酸之比的能力被认为可以控制细胞膜的结构完整性和流动性，从而在细胞生长，分化和细胞凋亡中起重要作用。我们观察到在4-HPR诱导的凋亡过程中，人RPE细胞中的SCD蛋白表达和酶活性降低。我们正在通过生化测定和对培养的RPE细胞的微阵列分析来研究通过SCD的抑制剂抑制4HPR诱导的人RPE细胞的凋亡。 我们已经修改并确定了与SCD途径有关的几种脂质代谢产物及其在细胞生长，分化和凋亡中的作用的HPLC和质谱测定法。 2）我们一直在与Chandra Nagineni和John Hooks（Nei-Li）合作，继续进行miRNA在调节成年人类视网膜上皮（HRPE）细胞的炎症反应中的作用。我们较早的工作表明，炎性细胞因子（IFN-GAMMA + TNF-ALPHA + IL-1BETA）可以显着增加HRPE细胞中miR-155表达的表达。尽管已知IFN-gamma通过激活JAK/STAT信号传导途径来调节基因表达，但该途径在调节miR-155表达中的作用尚未阐明。因此，我们研究了JAK/STAT途径在通过炎症细胞因子中介导HRPE细胞中miR-155表达的调节中的作用。我们在BIC/miR-155启动子的近端区域中检测到了两个推定的STAT1结合元件。电泳迁移率转移测定表明，与对照细胞相比，由处理细胞制备的核提取物中存在蛋白质与这两个元素的结合增加。炎症细胞因子的miR-155表达增加也与STAT1激活的增加有关。 JAK/Stat途径的抑制剂JAK抑制剂1有效地阻断了STAT1激活的增加，与STAT1元素的蛋白质结合和miR-155表达的增加。因此，我们的结果清楚地表明，炎性细胞因子通过直接激活JAK/STAT信号通路来增加人类视网膜色素上皮细胞中的miR-155。因此，通过JAK/STAT信号通路对miR-155表达的调节可能在导致AMD或其他视网膜退行性疾病的炎症过程中起重要作用。 3）在过去一年发表的两篇论文中反映了一项与翼状g相关过程的多LAB协作调查的结论。 翼状g是一种视力障碍的纤维血管病变，可在角膜表面生长，并与阳光暴露有关。我们使用了未归一化的未放大翼状cDNA文库的表达序列标签分析，以检查分离的翼粒的转录库，并鉴定组织起源和细胞迁移的标记基因。通过在正常眼眼表面和pterygium中的免疫荧光定位来验证所选基因的表达。来自pterygium的最丰富的互补DNA包括簇蛋白，角蛋白13（KRT13）和4（KRT4），S100A9/calgranulin b，以及精子/精子N1-乙酰转移酶（SAT1）。存在结膜（例如角蛋白13/4和AQP3）和角膜上皮（例如角蛋白12/3和AQP5）的标记。 其中几个基因在切除的翼状a和s100a9和sat1中表达最多的基因在细胞迁移中起作用。 SAT1通过控制多胺水平发挥作用。 ipenspm是一种多胺类似物，显示出降低pterygium饮食中迁移的显着能力。角蛋白和其他标志物在翼状a和其他标记最紧密的表达模式类似于结膜和边缘细胞的表达模式。存在一些角膜标记，尤其是KRT12，但比等效结膜标记的水平较低。我们的数据与从结膜和边缘样细胞迁移到角膜上皮形成的翼状二颗模型一致。鉴定在细胞迁移中作用的基因表明潜在的治疗靶标。特别是，多胺类似物减少pterygium的原发性培养物的迁移的能力提出了一种减缓翼状生长的方法。我们还发现，强力霉素会导致翼状鼠模型的消退。