Human retinal pigment epithelium proliferation, migration and fluid transport

人视网膜色素上皮增殖、迁移和液体运输

• 批准号: 7734635
• 负责人: sheldon s miller
• 金额: $ 42.51万
• 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/7734635
• 关键词: Affect; Apical; Apoptosis; Back; Bathing; Binding; Cell Proliferation; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoskeleton; Epidermal Growth Factor; Epithelial; Eye; Fibroblast Growth Factor 2; Functional disorder; Growth Factor; Health; Hour; Human; IRF2 gene; In Vitro; Inflammation; Inflammatory; Interferon Regulatory Factor 1; Interferon Type II; Interferon-alpha; Interferon-beta; Interferons; JAK3 gene; Liquid substance; Localized; MAP Kinase Gene; MAPK14 gene; Measurable; Mediating; Mediator of activation protein; Mitogen-Activated Protein Kinase Inhibitor; Neural Retina; Pathology; Pathway interactions; Phosphorylation; Photoreceptors; Platelet-Derived Growth Factor; Protein Isoforms; Protein Kinase A Inhibitor; Proteins; Receptor Activation; Retinal; Retinal Diseases; Retinal Pigments; Role; SB 203580; Structure of retinal pigment epithelium; Tight Junctions; Tissues; Vision; absorption; apical membrane; basolateral membrane; cell motility; cytokine; fetal; in vivo; inhibitor/antagonist; interferon gamma receptor; interferon gamma receptors; migration; monolayer; platelet-derived growth factor BB; platelet-derived growth factor C; response; therapeutic target; tyrphostin AG-490

PDGF-C and -D are the major isoforms expressed in human retinal pigment epithelium (RPE). Functionally active PDGFR-α and -β are mainly expressed at the apical membrane. PDGF-BB, -CC, -DD significantly increased proliferation while PDGF-BB, -AB and -DD significantly increased cell migration, suggesting a critical role in RPE pathophysiology. A pro-inflammatory cytokine mixture (TNFα/IL-1β/IFNγ) abrogated PDGF-induced proliferation and migration by inducing apoptosis, and by disrupting the cytoskeleton and tight junctions. Interferon gamma (IFNg) is one of the most important inflammatory mediators which are up-regulated in many retinal diseases. These sight threatening retinal diseases occur in the back of the eye; they mainly affect photoreceptors and the closely adjacent retinal pigment epithelial cells, which serve to protect the health and integrity of the neural retina. Functionally active IFNg receptor is mainly localized at the basolateral membrane of human fetal retinal pigment epithelium (hfRPE). The activation of this receptor significantly inhibits basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) induced RPE migration and proliferation. In contrast, type I IFN (IFN alpha and IFN beta) did not affect RPE proliferation. The inhibitory effects of IFNg on hfRPE were significantly blocked by JAK inhibitor I and by AG490 , but not by JAK3 inhibitor. In addition, activation of this JAK/STAT pathway up-regulates interferon regulatory factor 1 (IRF-1), but has no effect on IRF-2 and ICSBP/IRF-8. Interestingly, IFNg significantly stimulated the proliferation of cells from adjacent choroidal tissue. Addition of IFNg to the basal, but not the apical bath, significantly increased fluid transport (JV) across hfRPE monolayer. The IFNg induced JV increase was significantly blocked by a specific inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR) (CFTRinh-172). CFTR protein is expressed and mainly localized on the basolateral membrane of hfRPE. IFNg induced JV increase was blocked by pretreatment with cyclohexamide (4 or 24 hours). IFNg had no measurable effects on intracellular cAMP or Ca2+ levels. IFNg did however increase the phosphorylation of P38 MAPK. In addition, the IFNg induced JV increase was significantly reduced by the addition of P38 MAPK inhibitors, SB203580 and SB202190. A further JV decrease was produced by the subsequent addition of CFTRinh-172. Although IFNg has no measurable effect on intracellular cAMP, IFNg stimulated Jv increase was significantly inhibited by specific protein kinase A (PKA) inhibitor (H-89) and Rp-8-Br-cAMPS, which competitive blocked cAMP binding to PKA and blocked the effect of cAMP. We conclude that IFNg inhibits RPE migration and proliferation, activates CFTR-dependent fluid absorption across RPE in vitro and in vivo, and that JAK/STAT1, IRF-1, P38 MAPK and PKA are all involved in mediating these responses. These finding suggest several therapeutic targets for treating proliferative retinal diseases and removing the fluid accumulation in the subretinal space that occurs following many retinal pathologies.

PDGF-C和PDGF-D是人视网膜色素上皮细胞（RPE）中表达的主要亚型。功能活性PDGFR-和-主要在顶膜表达。PDGF-BB、-CC、-DD显著增加增殖，而PDGF-BB、-AB和-DD显著增加细胞迁移，表明在RPE病理生理学中的关键作用。促炎细胞因子混合物（TNF/IL-1/IFN）通过诱导细胞凋亡和破坏细胞骨架和紧密连接来消除PDGF诱导的增殖和迁移。 干扰素γ（Interferon gamma，IFNg）是一种重要的炎症介质，在多种视网膜疾病中表达上调。这些威胁视力的视网膜疾病发生在眼睛的后部;它们主要影响光感受器和紧密相邻的视网膜色素上皮细胞，其用于保护神经视网膜的健康和完整性。 IFNg受体主要定位于人胎儿视网膜色素上皮（hfRPE）的基底外侧膜。该受体的活化显著抑制碱性成纤维细胞生长因子（bFGF）、血小板衍生生长因子（PDGF）和表皮生长因子（EGF）诱导的RPE迁移和增殖。相反，I型IFN（IFN α和IFN β）不影响RPE增殖。 IFNg对hfRPE的抑制作用可被JAK抑制剂I和AG 490阻断，但不被JAK 3抑制剂阻断。此外，该JAK/STAT通路的激活上调干扰素调节因子1（IRF-1），但对IRF-2和ICSBP/IRF-8没有影响。有趣的是，IFNg显著刺激来自邻近脉络膜组织的细胞增殖。 将IFNg添加至基底浴而非顶端浴显著增加了穿过hfRPE单层的流体转运（JV）。IFNg诱导的JV增加被囊性纤维化跨膜传导调节因子（CFTR）的特异性抑制剂（CFTRinh-172）显著阻断。CFTR蛋白表达并主要定位于hfRPE的基底外侧膜上。IFNg诱导的JV增加通过用环己脲预处理（4或24小时）来阻断。 IFNg对细胞内cAMP或Ca 2+水平没有可测量的影响。然而，IFNg确实增加了P38 MAPK的磷酸化。此外，IFNg诱导的JV增加通过添加P38 MAPK抑制剂SB 203580和SB 202190而显著降低。随后添加CFTRinh-172导致JV进一步降低。尽管IFNg对细胞内cAMP无明显影响，但特异性蛋白激酶A（PKA）抑制剂（H-89）和Rp-8-Br-cAMPS可明显抑制IFNg刺激的Jv增加，竞争性阻断cAMP与PKA的结合，阻断cAMP的作用。我们的结论是，IFNg抑制RPE迁移和增殖，激活CFTR依赖的液体吸收在体外和体内的RPE，JAK/STAT 1，IRF-1，P38 MAPK和PKA都参与介导这些反应。这些发现提示了用于治疗增殖性视网膜疾病和去除在许多视网膜病变之后发生的视网膜下腔中的流体积聚的几种治疗靶点。