Cellular Interactions with Thrombospondin

细胞与血小板反应蛋白的相互作用

• 批准号: 10926575
• 负责人: david d roberts
• 金额: $ 141.13万
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 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/10926575
• 关键词: 7-methylguanosine; Acute; Alkylation; Angiogenesis Inhibition; Angiogenesis Inhibitors; Anthracycline; Autophagocytosis; Blocking Antibodies; Blood Platelets; Blood Pressure; Blood flow; Bone Marrow; CD47 gene; CD8-Positive T-Lymphocytes; CD81 gene; Cell Line; Cell Surface Receptors; Cell Survival; Cell membrane; Cells; Co-Immunoprecipitations; Code; Complex; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Cytoplasm; DNA Repair; Data; Eukaryotic Initiation Factor-4E; Extracellular Matrix; Family suidae; Genes; Genetic Transcription; Glycoproteins; Growth; Hemostatic function; Image; Integrins; Ionizing radiation; Irradiated tumor; Ischemia; Jurkat Cells; KDR gene; Knockout Mice; Lateral; Macrophage; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Messenger RNA; Metabolism; MicroRNAs; Mus; Myocardium; NOS3 gene; Natural Killer Cells; Nitric Oxide; Non-Malignant; Nuclear; Nuclear Export; Null Lymphocytes; PC3 cell line; Play; Precursor T-Lymphoblast; Prostate carcinoma; Proteins; RNA; Radiation Injuries; Rattus; Receptor Signaling; Recovery; Regulation; Reperfusion Injury; Reporting; Role; Running; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Smooth Muscle Myocytes; Soluble Guanylate Cyclase; Stromal Cells; T-Lymphocyte; Therapeutic; Thrombospondin 1; Thrombospondins; Tissue Survival; Tissues; Transgenic Mice; Tumor Immunity; UBQLN1 gene; Untranslated RNA; Vascular Smooth Muscle; adaptive immune response; angiogenesis; antagonist; anti-cancer; anti-tumor immune response; c-myc Genes; cancer cell; cancer immunotherapy; cancer stem cell; cell type; cytotoxic; exportin 1 protein; extracellular vesicles; immune checkpoint; inhibitor; ischemic injury; leptomycin B; malignant breast neoplasm; mouse model; neoplastic cell; novel; particle; preservation; radiation resistance; receptor; response; self-renewal; soft tissue; stem cells; therapeutic target; tissue culture; tissue stress; trafficking; transcription factor; transcriptomics; treatment response; tumor; tumor progression; tumor xenograft; vesicular release

CD47 is a marker of self and a signaling receptor for thrombospondin-1 that is also a component of extracellular vesicles (EVs) released by various cell types. Previous studies identified CD47-dependent functional effects of T cell EVs on target cells, mediated by delivery of their RNA contents, and enrichment of specific subsets of coding and noncoding RNAs in CD47+ EVs. Mass spectrometry was employed here to identify potential mechanisms by which CD47 regulates the trafficking of specific RNAs to EVs. Specific interactions of CD47 and its cytoplasmic adapter ubiquilin-1 with components of the exportin-1/Ran nuclear export complex were identified and confirmed by coimmunoprecipitation. Exportin-1 is known to regulate nuclear to cytoplasmic trafficking of 5'-7-methylguanosine (m7G)-modified microRNAs and mRNAs that interact with its cargo protein EIF4E. Interaction with CD47 was inhibited following alkylation of exportin-1 at Cys528 by its covalent inhibitor leptomycin B. Leptomycin B increased levels of m7G-modified RNAs, and their association with exportin-1 in EVs released from wild type but not CD47-deficient cells. In addition to perturbing nuclear to cytoplasmic transport, transcriptomic analyses of EVs released by wild type and CD47-deficient Jurkat T cells revealed a global CD47-dependent enrichment of m7G-modified microRNAs and mRNAs in EVs released by CD47-deficient cells. Correspondingly, decreasing CD47 expression in wild type cells or treatment with thrombospondin-1 enhanced levels of specific m7G-modified RNAs released in EVs, and re-expressing CD47 in CD47-deficient T cells decreased their levels. Therefore, CD47 signaling limits the trafficking of m7G-modified RNAs to EVs through physical interactions with the exportin-1/Ran transport complex. CD47 regulates the trafficking of specific coding and noncoding RNAs into extracellular vesicles (EVs), and the RNA contents of CD47+ EVs differ from that of CD63+ EVs released by the same cells. Single particle interferometric reflectance imaging sensing combined with immunofluorescent imaging was used to analyze the colocalization of tetraspanins, integrins, and CD47 on EVs produced by wild type and CD47-deficient Jurkat T lymphoblast and PC3 prostate carcinoma cell lines. On Jurkat cell-derived EVs, beta-1 and alpha-4 integrin subunits colocalized predominantly with CD47 and CD81 but not with CD63 and CD9, conserving the known lateral interactions between these proteins in the plasma membrane. Although PC3 cell-derived EVs lacked detectable alpha-4 integrin, specific association of CD81 with beta-1 and CD47 was preserved. Loss of CD47 expression in Jurkat cells significantly reduced beta-1 and alpha-4 levels on EVs produced by these cells while elevating CD9+ , CD63+ , and CD81+ EVs. In contrast, loss of CD47 in PC3 cells decreased the abundance of CD63+ and CD81+ EVs. These data establish that CD47+ EVs are mostly distinct from EVs bearing the tetraspanins CD63 and CD9, but CD47 also indirectly regulates the abundance of EVs bearing these non-interacting tetraspanins via mechanisms that remain to be determined.

CD47是自我的标记，也是血小板传播1的信号受体，它也是各种细胞类型释放的细胞外囊泡（EV）的组成部分。先前的研究确定了T细胞EVS对靶细胞的CD47依赖性功能效应，通过递送其RNA含量介导，以及在CD47+ EV中富集编码和非编码RNA的特定子集。这里采用了质谱法来确定CD47调节特定RNA向电动汽车的运输的潜在机制。通过共免疫沉淀确定并确认了CD47及其细胞质适配器泛素泛素-1与Exportin-1/RAN核出口复合物的成分的特异性相互作用。已知Exportin-1会调节与5'-7-甲基鸟苷（M7G）改性的microRNA和MRNA相互作用的核质量运输，这些核蛋白（M7G）与其货物蛋白EIF4E相互作用。在CYS528的Exportin-1烷基化在CYS528处的烷基化抑制剂瘦霉素B.钩霉素B的相互作用增加了M7G修饰的RNA水平，并且它们与野生型但不是CD47缺乏细胞的EVS中的EVS中的EVS中的相关性。除了扰动核对细胞质转运外，野生型和CD47缺乏的Jurkat T细胞对电动汽车的转录组分析表明，CD47缺乏细胞释放的M7G修饰的microRNA和MRNA的全球CD47依赖性富集M7G修饰的microRNA和mRNA。相应地，野生型细胞中的CD47表达降低或用血小板传播1的处理增强了EV中释放的特定M7G修饰的RNA水平，并在CD47缺陷型T细胞中重新表达CD47降低了其水平。因此，CD47信号传导通过与Exportin-1/RAN运输复合物的物理相互作用将M7G修饰的RNA的运输限制为电动汽车。 CD47调节特定编码和非编码RNA的运输到细胞外囊泡（EV）中，CD47+ EV的RNA含量与同一细胞释放的CD63+ EV的RNA含量不同。与免疫荧光成像结合使用的单个颗粒干涉反射率成像被用于分析由野生型和CD47缺乏的Jurkat T淋巴细胞和PC3 PC3前列腺癌细胞线产生的EVS上的四叠蛋白，整联蛋白和CD47的共定位。在Jurkat细胞衍生的EV上，Beta-1和Alpha-4整合蛋白亚基主要与CD47和CD81共定位，而与CD63和CD9共定位，并保留了质膜中这些蛋白质之间已知的横向相互作用。尽管缺乏可检测到的α-4整联蛋白的PC3细胞衍生的EVS，但CD81与β-1和CD47的特异性关联得到了保留。在Jurkat细胞中CD47表达的丧失可显着降低这些细胞产生的EV上的β-1和α-4水平，同时升高CD9+，CD63+和CD81+ EV。相反，PC3细胞中CD47的损失降低了CD63+和CD81+ EV的丰度。这些数据表明，CD47+ EV大多与含有四跨果蛋白CD63和CD9的EV不同，但是CD47还间接地调节了具有这些非相互作用的四翼烷蛋白的EVS的丰度，这些电动汽车通过仍有待确定的机制来调节。

项目成果

Endogenous thrombospondin-1 regulates leukocyte recruitment and activation and accelerates death from systemic candidiasis.

• DOI: 10.1371/journal.pone.0048775
• 发表时间: 2012
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Martin-Manso G;Navarathna DH;Galli S;Soto-Pantoja DR;Kuznetsova SA;Tsokos M;Roberts DD
• 通讯作者: Roberts DD

Blockade of CD47 increases survival of mice exposed to lethal total body irradiation.

• DOI: 10.1038/srep01038
• 发表时间: 2013
• 期刊: SCIENTIFIC REPORTS
• 影响因子: 4.6
• 作者: Soto-Pantoja, David R.;Ridnour, Lisa A.;Wink, David A.;Roberts, David D.
• 通讯作者: Roberts, David D.

Secreted Thrombospondin-1 Regulates Macrophage Interleukin-1β Production and Activation through CD47.

• DOI: 10.1038/srep19684
• 发表时间: 2016-01-27
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Stein EV;Miller TW;Ivins-O'Keefe K;Kaur S;Roberts DD
• 通讯作者: Roberts DD

CD47-dependent immunomodulatory and angiogenic activities of extracellular vesicles produced by T cells.

• DOI: 10.1016/j.matbio.2014.05.007
• 发表时间: 2014-07
• 期刊: Matrix biology : journal of the International Society for Matrix Biology
• 作者: Kaur S;Singh SP;Elkahloun AG;Wu W;Abu-Asab MS;Roberts DD
• 通讯作者: Roberts DD

Inhibitory signaling through signal regulatory protein-α is not sufficient to explain the antitumor activities of CD47 antibodies.

通过信号调节蛋白-α 的抑制信号不足以解释 CD47 抗体的抗肿瘤活性。

• DOI: 10.1073/pnas.1205441109
• 发表时间: 2012
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Soto-Pantoja,DavidR;Miller,ThomasW;Frazier,WilliamA;Roberts,DavidD
• 通讯作者: Roberts,DavidD