Signaling in the retina and retinal pigment epithelium

过去一年，我们在以下方面取得了进展： 1)继续努力确定在4-HPR诱导的人RPE(ARPE-19)细胞神经分化中Igfbp5表达下调，我们表明Igfbp5表达下调似乎是由于神经元分化，而不是人RPE细胞神经分化的先决条件(Samuel等人，J.Cell Physiol)。224：827-836,2010)。我们发现，MEK1/2的抑制剂U0126抑制了Igfbp5的下调，表明MAPK通路参与了这一过程。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Igfbp5表达下调和RPE细胞的神经分化。有趣的是，在凝胶位移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，在报告实验中，人Igfbp5启动子的C/EBP反应元件的缺失显著降低了启动子的基础活性，并取消了其对4HPR处理的反应性，表明Igfbp5的表达受C/EBP的调控。我们还继续努力确定硬脂酰辅酶A脱饱和酶(SCD)在RPE细胞死亡中的作用，SCD是不饱和脂肪酸合成中的限速酶。SCD通过调节饱和脂肪酸和单不饱和脂肪酸的比例，控制细胞膜的结构完整性和流动性，从而在细胞的生长、分化和凋亡中发挥重要作用。我们观察到，在4-HPR诱导人RPE细胞凋亡的过程中，SCD蛋白表达和酶活性降低。我们正在通过生化分析和培养的RPE细胞芯片分析来研究SCD抑制剂对4HPR诱导的人RPE细胞凋亡的抑制作用。我们已经改进和建立了几种与SCD途径及其在细胞生长、分化和凋亡中的作用有关的脂代谢产物的高效液相色谱和质谱学分析方法。 2)我们继续与Chandra Nagineni和John Hooks(Nei-Li)合作，研究miRNA在调节成人视网膜色素上皮(HRPE)细胞炎症反应中的作用。我们早期的工作表明，炎性细胞因子(干扰素-γ、肿瘤坏死因子-α、IL-1β)能显着增加hRPE细胞miR-155的表达。虽然已知干扰素-γ通过激活JAK/STAT信号通路来调节基因表达，但该通路在调节miR-155表达中的作用尚不清楚。因此，我们研究了JAK/STAT通路在炎性细胞因子调节hRPE细胞miR-155表达中的作用。我们在BIC/miR-155启动子的近端区域检测到两个可能的STAT1结合元件。电泳迁移率改变分析表明，与对照细胞相比，处理细胞制备的核提取液中与这两种元素的结合蛋白增加。炎性细胞因子增加miR-155的表达还与STAT1的激活增加有关。JAK/STAT通路的抑制剂JAK Inhibitor 1有效地阻断了STAT1的激活、与STAT1元件的蛋白结合和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，增加了人视网膜色素上皮细胞miR-155的表达。因此，JAK/STAT信号通路对miR-155表达的调控可能在导致AMD或其他视网膜退行性疾病的炎症过程中发挥重要作用。 3)在过去一年发表的两篇论文中，反映了一项多实验室合作研究的结论，该研究描述了与翼状胬肉相关的过程。翼状胬肉是一种视力受损的纤维血管病变，生长在角膜表面，与阳光照射有关。我们利用一个非标准化的未扩增翼状胬肉cDNA文库的表达序列标签分析来检测分离的翼状胬肉的转录谱系，并寻找组织起源和细胞迁移的标记基因。免疫荧光定位证实了所选基因在正常眼表面和翼状胬肉中的表达。翼状胬肉中最丰富的互补DNA包括聚集素、角蛋白13(Krt13)和角蛋白4(Krt4)、S100A9/钙颗粒蛋白B和亚精胺/精胺N1-乙酰转移酶(SAT1)。结膜(如角蛋白13/4和AQP3)和角膜上皮(如角蛋白12/3和AQP5)的标记物都存在。在切除的翼状胬肉中表达最丰富的几个基因，特别是S100A9和SAT1，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，显示出显著的减少翼状胬肉原代培养细胞迁移的能力。角蛋白和其他标志物在翼状胬肉中的表达模式与结膜和角膜缘细胞最为相似；存在一些角膜标志物，特别是Krt12，但水平低于同等的结膜标志物。我们的数据与翼状胬肉的模型是一致的，该模型是由结膜和角膜缘样细胞向角膜上皮细胞迁移形成的。识别在细胞迁移中起作用的基因暗示了潜在的治疗靶点。特别是，多胺类似物在翼状胬肉原代培养中减少迁移的能力提供了一种减缓翼状胬肉生长的可能方法。我们还发现，多西环素可导致翼状胬肉小鼠模型的消退。

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.