Cellular Interactions with Thrombospondin

细胞与血小板反应蛋白的相互作用

基本信息

批准号：
8763691
负责人：
david d roberts
金额：
$ 94.58万
依托单位：
DIVISION OF CLINICAL SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/8763691
关键词：
Acute Adverse effects Angiogenesis Inhibition Angiogenesis Inhibitors Autophagocytosis Autophagosome Blood Cell Count Blood Platelets Blood Pressure Blood Vessels Blood flow Bone Marrow CD47 Antigen CD47 gene Cardiovascular Physiology Cell Death Cell Line Cell Surface Receptors Cell Survival Cells Cessation of life Cyclic GMP Cyclic GMP-Dependent Protein Kinases Data Differentiation and Growth Dose Electron Microscopy Endothelial Cells Exhibits Exposure to Extracellular Matrix Family suidae Gene Expression Genes Glycoproteins Hematopoietic Hemostatic function Human Immune response Injury Investigational Drugs Ionizing radiation Ischemia Knockout Mice Ligands Ligation Malignant Neoplasms Mediating Membrane Messenger RNA Molecular Mus Nitric Oxide Normal tissue morphology Nuclear Accidents Null Lymphocytes Pathway interactions Peripheral Physiological Play Proteins Radiation Radiation Injuries Radiation Syndromes Radioprotection Radioresistance Rattus Recovery Regulation Relative (related person)Reperfusion Injury Reporting Role Route Signal Pathway Signal Transduction Signal Transduction Pathway Small Interfering RNA Smooth Muscle Myocytes Soluble Guanylate Cyclase Stem cells Stress Stromal Cells T-Lymphocyte THBS1 gene Terrorism Therapeutic Thrombospondin 1 Tissue Survival Tissues Transgenic Mice Tumor Immunity Vascular Endothelial Growth Factor Receptor Vesicle Whole-Body Irradiation angiogenesis antiangiogenesis therapy base c-myc Genes cell type gastrointestinal gene discovery human NOS3 protein in vivo inhibition of autophagy mouse model neoplastic cell peptide analog prevent protein aminoacid sequence receptor response self-renewal soft tissue therapeutic angiogenesis tissue culture transcription factor tumor tumor progression tumor xenograft

项目摘要

Accidental or therapeutic exposure to ionizing radiation has severe physiological consequences and can result in cell death. We previously demonstrated that deficiency or blockade of the ubiquitously expressed receptor CD47 results in remarkable cell and tissue protection against ischemic and radiation stress. Antagonists of CD47 or its ligand THBS1/thrombospondin 1 enhance cell survival and preserve their proliferative capacity. However the signaling pathways that mediate this cell-autonomous radioprotection are unclear. We now report a marked increase in autophagy in irradiated T-cells and endothelial cells lacking CD47. Irradiated T cells lacking CD47 exhibit significant increases in formation of autophagosomes comprising double-membrane vesicles visualized by electron microscopy and numbers of MAP1LC3A/B(+) puncta. Moreover, we observed significant increases in BECN1, ATG5, ATG7 and a reduction in SQSTM1/p62 expression relative to irradiated wild-type T cells. We observed similar increases in autophagy gene expression in mice resulting from blockade of CD47 in combination with total body radiation. Pharmacological or siRNA-mediated inhibition of autophagy selectively sensitized CD47-deficient cells to radiation, indicating that enhanced autophagy is necessary for the prosurvival response to CD47 blockade. Moreover, re-expression of CD47 in CD47-deficient T cells sensitized these cells to death by ionizing radiation and reversed the increase in autophagic flux associated with survival. This study indicates that CD47 deficiency confers cell survival through the activation of autophagic flux and identifies CD47 blockade as a pharmacological route to modulate autophagy for protecting tissue from radiation injury. Accidental or therapeutic total body exposure to ionizing radiation has profound pathophysiological consequences including acute radiation syndrome. Currently only investigational drugs are available in case of radiological or nuclear accidents or terrorism. Lack of selective radioprotectants for normal tissues also limits the therapeutic doses that can be delivered to treat cancers. CD47 is a receptor for the secreted protein thrombospondin-1. Blockade of thrombospondin-1 or CD47 provides local radioprotection of soft tissues and bone marrow. We now report that suppression of CD47 using an antisense morpholino increases survival of mice exposed to lethal total body irradiation. Increased survival is associated with increased peripheral circulating blood cell counts and increased proliferative capacity of bone marrow derived cells. Moreover, CD47 blockade decreased cell death while inducing a protective autophagy response in radiosensitive gastrointestinal tissues. Thus, CD47 is a new target for radiomitigation that prevents both hematopoietic and gastrointestinal radiation syndromes. Signaling through the thrombospondin-1 receptor CD47 broadly limits cell and tissue survival of stress, but the molecular mechanisms are incompletely understood. We now show that loss of CD47 permits sustained proliferation of primary murine endothelial cells, increases asymmetric division, and enables these cells to spontaneously reprogram to form multipotent embryoid body-like clusters. c-Myc, Klf4, Oct4, and Sox2 expression is elevated in CD47-null endothelial cells, in several tissues of CD47- and thrombospondin-1-null mice, and in a human T cell line lacking CD47. CD47 knockdown acutely increases mRNA levels of c-Myc and other stem cell transcription factors in cells and in vivo, whereas CD47 ligation by thrombospondin-1 suppresses c-Myc expression. The inhibitory effects of increasing CD47 levels can be overcome by maintaining c-Myc expression and are absent in cells with dysregulated c-Myc. Thus, CD47 antagonists enable cell self-renewal and reprogramming by overcoming negative regulation of c-Myc and other stem cell transcription factors.

意外或治疗暴露于电离辐射会带来严重的生理后果，并可能导致细胞死亡。我们先前证明，普遍表达的受体CD47的缺乏或阻断导致细胞和组织保护对缺血性和辐射应力的显着保护。 CD47或其配体THBS1/血小板素1的拮抗剂1增强细胞存活并保持其增殖能力。但是，介导该细胞自主放射保护的介导的信号传导途径尚不清楚。现在，我们报告说，缺乏CD47的辐照的T细胞和内皮细胞的自噬显着增加。缺乏CD47的受照射的T细胞在形成的自噬体的形成显着增加，其中包括通过电子显微镜观察到的双膜囊泡和MAP1LC3A/B（+）点的数量。此外，我们观察到相对于受辐照的野生型T细胞的BECN1，ATG5，ATG7，ATG7，ATG7的显着增加以及SQSTM1/p62表达的降低。我们观察到与总体辐射结合使用CD47导致的小鼠自噬基因表达的相似增加。药理学或siRNA介导的自噬选择性敏化CD47缺陷细胞对辐射的抑制作用，表明增强的自噬对于Proservival对CD47封锁的反应是必需的。此外，CD47在CD47缺陷型T细胞中的重新表达通过电离辐射使这些细胞死亡，并逆转与生存有关的自噬通量的增加。这项研究表明，CD47缺乏通过激活自噬通量来赋予细胞存活，并确定CD47封锁是调节自噬以保护组织免受辐射损伤的药理途径。意外或治疗性全身暴露于电离辐射中具有深远的病理生理后果，包括急性辐射综合征。目前，仅在放射学或核事故或恐怖主义的情况下，只能使用研究药物。缺乏正常组织的选择性放射保护剂也限制了可以递送以治疗癌症的治疗剂量。 CD47是分泌蛋白血小板蛋白1的受体。血小板素-1或CD47的阻断提供了软组织和骨髓的局部辐射保护。现在，我们报告说，使用反义morpholino的CD47抑制会增加暴露于致命的总体辐照的小鼠的存活率。增加的生存率与外周循环血细胞计数增加以及骨髓衍生细胞的增殖能力增加有关。此外，CD47阻断减少了细胞死亡，同时诱导了放射敏感性胃肠道组织的保护性自噬反应。因此，CD47是放射性措施的新目标，可防止造血和胃肠道辐射综合征。通过血小板素-1受体CD47的信号传导广泛地限制了应激的细胞和组织存活，但是分子机制却不完全了解。我们现在表明，CD47的损失允许原代鼠内皮细胞的持续增殖，增加不对称的分裂，并使这些细胞能够自发地重编程形成多型胚胎身体样簇。 C-Myc，KLF4，OCT4和SOX2表达在CD47无效的内皮细胞中，CD47和血小板传播1-无效小鼠的几个组织以及缺乏CD47的人类T细胞系中升高。 CD47敲低急性增加细胞和体内干细胞转录因子的mRNA水平和其他干细胞转录因子的水平，而血小板传播1通过CD47结扎抑制了C-MYC的表达。 CD47水平升高的抑制作用可以通过保持C-MYC表达来克服，并且在C-MYC失调的细胞中不存在。因此，CD47拮抗剂通过克服C-MYC和其他干细胞转录因子的负调控来实现细胞自我更新和重编程。