Signaling in the retina and retinal pigment epithelium

过去一年，我们在以下领域取得了进展： 1)鉴定人RPE（ARPE-19）细胞的4-HPR诱导的神经元分化中IGFBP 5表达的下调的努力继续进行，并且我们显示IGFBP 5表达的降低似乎是由于神经元分化，而不是人RPE细胞的神经元分化的先决条件（Samuel等人，224：827-836，2010）。我们发现IGFBP 5的下调被MEK 1/2的抑制剂U 0126抑制，表明MAPK通路的参与。转录因子C/EBP（CCAAT/enhancer binding proteins）的过表达抑制了4-HPR诱导的IGFBP 5表达下调和RPE细胞的神经元分化。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/EBP β与IGFBP 5启动子的结合。此外，从人IGFBP 5启动子中删除C/EBP反应元件显著降低了基础启动子活性，并在报告基因测定中消除了其对4 HPR处理的反应性，表明IGFBP 5的表达受C/EBP调节。我们还继续努力，以确定硬脂酰辅酶A去饱和酶（SCD），不饱和脂肪酸合成的限速酶，在RPE细胞死亡的作用。SCD通过其调节饱和脂肪酸与单不饱和脂肪酸的比率的能力被认为控制细胞膜的结构完整性和流动性，从而在细胞生长、分化和凋亡中起重要作用。我们观察到SCD蛋白表达和酶活性在4-HPR诱导的人RPE细胞凋亡过程中下降。我们正在通过培养的RPE细胞的生物化学测定和微阵列分析来研究SCD抑制剂对4 HPR诱导的人RPE细胞凋亡的抑制。 我们已经修改并建立了HPLC和质谱分析涉及SCD途径及其在细胞生长，分化和凋亡中的作用的几种脂质代谢产物。 2)我们与Chandra Nagineni和John Hooks（NEI-LI）合作，继续研究miRNA在调节成人视网膜色素上皮（HRPE）细胞炎症反应中的作用。我们的前期工作表明，炎症细胞因子（IFN-γ + TNF-α + IL-1 β）可以显著增加HRPE细胞中miR-155的表达。虽然已知IFN-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表达中的作用尚未阐明。因此，我们研究JAK/STAT通路在炎症因子介导HRPE细胞中miR-155表达调控中的作用。我们在BIC/miR-155启动子近端区域检测到两个可能的STAT 1结合元件。电泳迁移率变动分析表明，增加的蛋白质结合这两个元素是存在于从处理的细胞相比，对照细胞制备的核提取物。炎性细胞因子引起的miR-155表达的增加也与STAT 1活化的增加相关。STAT 1活化、蛋白与STAT 1元件结合和miR-155表达的增加被JAK抑制剂1（JAK/STAT途径的抑制剂）有效地阻断。因此，我们的结果清楚地表明，炎症细胞因子通过直接激活JAK/STAT信号通路增加人视网膜色素上皮细胞中miR-155的表达。因此，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发挥重要作用。 3)一项多实验室合作研究的结论描述了翼状胬肉相关过程的特征，反映在去年发表的2篇论文中。 翼状胬肉是一种损害视力的纤维血管病变，生长在角膜表面，与阳光照射有关。我们使用表达序列标签分析的非标准化的未扩增的翼状胬肉cDNA文库，以检查孤立的翼状胬肉的转录谱，并确定标记基因的组织起源和细胞迁移。通过免疫荧光定位在正常眼眼表和翼状胬肉中验证所选基因的表达。翼状胬肉中最丰富的互补DNA包括丛生蛋白、角蛋白13（Krt 13）和4（Krt 4）、S100 A9/钙颗粒蛋白B和亚精胺/精胺N1-乙酰转移酶（SAT 1）。结膜（如角蛋白13/4和AQP 3）和角膜上皮（如角蛋白12/3和AQP 5）的标志物均存在。 在切除的翼状胬肉中表达最丰富的几个基因，特别是S100 A9和SAT 1，在细胞迁移中起作用。SAT 1通过控制多胺水平发挥其作用。IPENSpm是一种多胺类似物，在翼状胬肉的原代培养中显示出显著的减少迁移的能力。角蛋白和其他标记物在翼状胬肉中的表达模式与结膜和角膜缘细胞的表达模式最相似;存在一些角膜标记物，特别是Krt 12，但水平低于等效结膜标记物。我们的数据与翼状胬肉的模型一致，翼状胬肉是由结膜样细胞和角膜缘样细胞迁移到角膜上皮形成的。鉴定在细胞迁移中起作用的基因提示了潜在的治疗靶点。特别是，多胺类似物减少翼状胬肉原代培养物迁移的能力提供了一种减缓翼状胬肉生长的可能方法。我们还发现强力霉素可引起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.