PHAGOCYTOSIS AND MERTK FAMILY OF THE RECEPTOR TYROSINE KINASE

受体酪氨酸激酶的吞噬作用和 MERTK 家族

• 批准号: 7959958
• 负责人: Qingxian Lu
• 金额: $ 8.83万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7959958
• 关键词: Affect; Birth; Cell Differentiation process; Cells; Cessation of life; Circadian Rhythms; Computer Retrieval of Information on Scientific Projects Database; DNA Sequence Rearrangement; Defect; Development; Developmental Process; Disease; Distal; Ectopic Expression; Electrophysiology (science); Epithelium; Eye Development; Family; Funding; Gene Expression Profiling; Genes; Grant; Homeostasis; Human; In Vitro; Inherited; Institution; Investigation; Knockout Mice; MERTK gene; Measurement; Mediating; Messenger RNA; Metaphase; Mitosis; Modeling; Molecular; Morphology; Mus; Mutant Strains Mice; Mutation; Natural Killer Cells; Organ; PTTG1 gene; Patients; Pattern; Phagocytes; Phagocytosis; Phenotype; Photoreceptors; Physiological; Pregnancy; Proteins; RNA Interference; Rattus; Receptor Protein-Tyrosine Kinases; Regulation; Research; Research Personnel; Resources; Retina; Retinal; Retinal Photoreceptors; Retinitis Pigmentosa; Rod Outer Segments; Role; Sister Chromatid; Source; Spermatogenesis; Structure; Surgeon; Testing; Time; United States National Institutes of Health; Vision; Visual; college; human MERTK protein; in vivo; macrophage; mutant; null mutation; overexpression; photoreceptor degeneration; postnatal; prevent; receptor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. While vertebrate eye development and functional differentiation is initiated in mid gestation, it is of note that the retina is perhaps the last organ to complete functional development. Most retinal differentiation occurs postnatally, and in the mouse retinal development is not completed until two weeks after birth. Thus, developmental defects in the retina are not evident functionally until significantly after birth. The retinal pigmenl epithelium (RPE) cells are highly polarized and they become tightly associated with the developing retinal photoreceptor cells. Once the retina becomes functional, the RPE is essential for phagocytic clearance of the spent photoreceptor distal outer segment (OS) tips; this function is essential to maintain photoreceptor homeostasis. The RPE develops well before the retina, but developmental defects in the RPE do not become evident until the postnatal period when the retina becomes functional. Developmental defects in RPE phagocytosis of the OS leads to photoreceptor death and the postnatal disease retinitis pigmentosa (RP). The role of the RPE in rod OS turnover has been extensively studied, due to the availability of the Royal College of Surgeons (RCS) rat model, which displays an RP phenotype. The RPE cells in these rats carry an inherited defect in rod OS phagocytosis and the mutant gene is the MerTK receptor. The MerTK receptor tyrosine kinase is essential for RPE phagocytosis. MerTK null mutation causes photoreceptor degeneration not only in the RCS rat, but also in MerTK gene knockout mice and human RP patients. In addition, the MerTK mutation leads to general developmental defects in NK cell differentiation, spermatogenesis, and macrophage phagocytosis. To understand the molecular role of MerTK in phagocytosis in these developmental process, we performed gene expression profiling to identify downstream targets of MerTK. One gene known as PTTG was ectopically expressed with MerTK mutation in RPE and macrophages. Interestingly, PTTG regulates cytoskeletal changes in dividing cells, PTTG stabilizes cytoskeletal structures such as the spindle apparatus, which separates sister chromatids during mitosis. However, PTTG must be degraded for this cytoskeletal structure to be disassembled at the end of mitotic metaphase. Thus, PTTG is required for cytoskeletal changes to occur in the cell. We have now crossed the MerTK mutant mice into a PTTG heterozygous background to reduce ectopic expression of PTTG, and we have found that photoreceptor loss in the MerTK mutant is largely prevented. These results point toward ectopic expression of PTTG as a critical factor in the RPE defects in the MerTK mutant. We hypothesize that ectopic expression of PTTG in the MerTK mutant RPE prevents the cytoskeletal rearrangements required to initiate phagocytosis (in a fashion analogous to that seen in cytoskeletal rearrangement defects seen in mitosis when PTTG becomes overexpressed). We now focus this proposal on investigation of the potential role for PTTG ectopic expression in the MerTK mutant phenotypes, and how the PTTG affect MerTK-mediated phagocytosis. Specific Aim 1. Examination of developmental and circadian regulation of PTTG expression and its role in phagocytosis of photoreceptor OS in the MerTK-/- RPE cells in vivo. In this Specific Aim, we will investigate the developmental expression pattern and circadian regulation of PTTG in RPE cells at both mRNA and protein level. We will study the photoreceptor degeneration time course and determine if phagocytosis is restored in MerTK-/-PTTG+/- mice. Specific Aim 2. Investigate whether in vitro ectopic expression of PTTG leads to defects in RPE phagocytosis. We will ectopically PTTG in normal RPE cells to examine if it will cause RPE cell phagocytic defects. Conversely, we will knockdown PTTG expression by RNA interference in the RPE cells with MerTK mutation and investigate whether decreased PTTG expression is sufficient to restore phagocytosis. Specific Aim 3. Functional studies of the MerTK-/- and MerTK-/-PTTG+/- retina by visual test and ERG analysis. We will perform electrophysiology and functional vision measurements on the MerTK-/-PTTG+/- mice. These physiological measurements will be correlated with a detailed morphological analysis of retinal markers and ultrastructual morphology.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 虽然脊椎动物的眼睛发育和功能分化是在妊娠中期开始的，但值得注意的是，视网膜可能是完成功能发育的最后一个器官。 大多数视网膜分化发生在出生后，并且小鼠视网膜的发育直到出生后两周才完成。 因此，直到出生后，视网膜的发育缺陷在功能上才明显。 视网膜色素上皮（RPE）细胞高度极化，它们与发育中的视网膜感光细胞紧密相关。一旦视网膜恢复功能，RPE 对于吞噬清除废光感受器远端外节 (OS) 尖端至关重要；该功能对于维持光感受器稳态至关重要。 RPE 的发育早于视网膜，但 RPE 的发育缺陷直到出生后视网膜恢复功能时才变得明显。 RPE 吞噬 OS 的发育缺陷会导致光感受器死亡和产后疾病视网膜色素变性 (RP)。由于皇家外科医生学院 (RCS) 大鼠模型显示 RP 表型，RPE 在视杆细胞 OS 周转中的作用已得到广泛研究。这些大鼠的 RPE 细胞在 rod OS 吞噬功能方面存在遗传性缺陷，突变基因是 MerTK 受体。 MerTK 受体酪氨酸激酶对于 RPE 吞噬作用至关重要。 MerTK 无效突变不仅会导致 RCS 大鼠的光感受器变性，还会导致 MerTK 基因敲除小鼠和人类 RP 患者的光感受器变性。此外，MerTK突变导致NK细胞分化、精子发生和巨噬细胞吞噬作用方面的普遍发育缺陷。为了了解 MerTK 在这些发育过程中吞噬作用的分子作用，我们进行了基因表达谱分析，以确定 MerTK 的下游靶标。一种称为 PTTG 的基因在 RPE 和巨噬细胞中异位表达并伴有 MerTK 突变。有趣的是，PTTG 调节分裂细胞中的细胞骨架变化，PTTG 稳定细胞骨架结构，例如纺锤体，在有丝分裂期间分离姐妹染色单体。然而，PTTG 必须被降解，才能使这种细胞骨架结构在有丝分裂中期结束时分解。因此，PTTG 是细胞中发生细胞骨架变化所必需的。我们现在将 MerTK 突变小鼠与 PTTG 杂合背景杂交，以减少 PTTG 的异位表达，并且我们发现 MerTK 突变体中的光感受器损失在很大程度上得到了预防。这些结果表明 PTTG 的异位表达是 MerTK 突变体 RPE 缺陷的关键因素。我们假设 MerTK 突变体 RPE 中 PTTG 的异位表达阻止了启动吞噬作用所需的细胞骨架重排（其方式类似于当 PTTG 过度表达时在有丝分裂中看到的细胞骨架重排缺陷）。 我们现在重点研究 PTTG 异位表达在 MerTK 突变表型中的潜在作用，以及 PTTG 如何影响 MerTK 介导的吞噬作用。 具体目标 1. 检查 PTTG 表达的发育和昼夜节律调节及其在体内 MerTK-/- RPE 细胞中光感受器 OS 吞噬作用中的作用。 在这个具体目标中，我们将在 mRNA 和蛋白质水平上研究 RPE 细胞中 PTTG 的发育表达模式和昼夜节律调节。我们将研究光感受器变性的时间过程，并确定 MerTK-/-PTTG+/- 小鼠的吞噬作用是否恢复。 具体目标 2. 研究 PTTG 的体外异位表达是否导致 RPE 吞噬功能缺陷。 我们将对正常RPE细胞进行异位PTTG，检查是否会引起RPE细胞吞噬缺陷。相反，我们将通过 RNA 干扰敲低具有 MerTK 突变的 RPE 细胞中的 PTTG 表达，并研究降低的 PTTG 表达是否足以恢复吞噬功能。 具体目标 3. 通过视觉测试和 ERG 分析对 MerTK-/- 和 MerTK-/-PTTG+/- 视网膜进行功能研究。 我们将对 MerTK-/-PTTG+/- 小鼠进行电生理学和功能性视觉测​​量。这些生理测量将与视网膜标记物和超微结构形态的详细形态学分析相关。