Project description:Intercellular communication is critical for integrating complex signals in multicellular eukaryotes. Vascular endothelial cells and T lymphocytes closely interact during the recirculation and trans-endothelial migration of T cells. In addition to direct cell-cell contact, we show that T cell derived extracellular vesicles can interact with endothelial cells and modulate their cellular functions. Thrombospondin-1 and its receptor CD47 are expressed on exosomes/ectosomes derived from T cells, and these extracellular vesicles are internalized and modulate signaling in both T cells and endothelial cells. Extracellular vesicles released from cells expressing or lacking CD47 differentially regulate activation of T cells induced by engaging the T cell receptor. Similarly, T cell-derived extracellular vesicles modulate endothelial cell responses to vascular endothelial growth factor and tube formation in a CD47-dependent manner. Uptake of T cell derived extracellular vesicles by recipient endothelial cells globally alters gene expression in a CD47-dependent manner. CD47 also regulates the mRNA content of extracellular vesicles in a manner consistent with some of the resulting alterations in target endothelial cell gene expression. Therefore, the thrombospondin-1 receptor CD47 directly or indirectly regulates intercellular communication mediated by the transfer of extracellular vesicles between vascular cells. HuVEC cells were cocultured with exosomes derived either from Jurkat or JinB8 cells culture media. Each condition was done in triplicate. Also, Exosome RNA from Jurkat or JINB8 cells were compared to each other in triplicate.
Project description:Intercellular communication is critical for integrating complex signals in multicellular eukaryotes. Vascular endothelial cells and T lymphocytes closely interact during the recirculation and trans-endothelial migration of T cells. In addition to direct cell-cell contact, we show that T cell derived extracellular vesicles can interact with endothelial cells and modulate their cellular functions. Thrombospondin-1 and its receptor CD47 are expressed on exosomes/ectosomes derived from T cells, and these extracellular vesicles are internalized and modulate signaling in both T cells and endothelial cells. Extracellular vesicles released from cells expressing or lacking CD47 differentially regulate activation of T cells induced by engaging the T cell receptor. Similarly, T cell-derived extracellular vesicles modulate endothelial cell responses to vascular endothelial growth factor and tube formation in a CD47-dependent manner. Uptake of T cell derived extracellular vesicles by recipient endothelial cells globally alters gene expression in a CD47-dependent manner. CD47 also regulates the mRNA content of extracellular vesicles in a manner consistent with some of the resulting alterations in target endothelial cell gene expression. Therefore, the thrombospondin-1 receptor CD47 directly or indirectly regulates intercellular communication mediated by the transfer of extracellular vesicles between vascular cells. Treatment with B6H12 antibody inhibited co-immunoprecipitation of EGFR with CD47 and inhibited EGF-induced EGFR tyrosine phosphorylation. B6H12 treatment of bCSC also suppressed asymmetric cell division and cell proliferation and up-regulated caspase 3/7 activity. Correspondingly, caspase-7 cleavage in human breast cancers correlated with CD47 expression. Our data shows that B6H12 specifically targets bCSCs but not differentiated cancer cells, and this CD47 signaling is independent of SIRPα. Three replicates of each condition were generated. Three replicates of each MDA-231 attached cells (differentiated), MDA-231 in suspension cells (bCSC), MDA-231 in suspension cells (bCSC) treated with Control Antibody and MDA-231 in suspension cells (bCSC) treated with B6H12 Antibody.
Project description:CD47 is a marker of self and a signaling receptor for thrombospondin-1 that is also a membrane
component of extracellular vesicles (EVs) released by various cell types. Previous studies identified CD47-dependent functional effects of EVs on target cells, mediated by delivery of their RNA contents, and enrichment of specific subsets of coding and noncoding RNAs in CD47+ EVs. Here, transcriptomic analyses of EVs released by human and murine cells revealed CD47-dependent enrichment of capped microRNAs and mRNAs. Knockdown or loss of CD47 in wild type Jurkat T cells or treatment with thrombospondin-1 enhanced levels of specific capped-RNAs released in EVs, and reexpressing CD47 in null cells decreased their levels. Mass spectrometry and co-immunoprecipitation identified specific interactions of CD47 with components of the exportin-1/Ran nuclear export complex and its known cargo proteins and between the CD47 cytoplasmic adapter ubiquilin-1 and the exportin-1/Ran complex. Interaction with CD47 was inhibited following alkylation of exportin-1 at Cys528 by leptomycin B. Leptomycin B treatment increased levels of cap-dependent RNAs and their association with exportin-1 in EVs released from wild type but not CD47-deficient cells. These results indicate that CD47
regulates the trafficking of cap-dependent RNAs to EVs through physical interactions with the exportin-1/Ran transport complex. Within the files, lmj1038 represents the control pulldown from CD47- JinB8 cells whereas lmj1039 is the CD47+ pulldown from wild-type Jurkat cells.
Project description:Intercellular communication is critical for integrating complex signals in multicellular eukaryotes. Vascular endothelial cells and T lymphocytes closely interact during the recirculation and trans-endothelial migration of T cells. In addition to direct cell-cell contact, we show that T cell derived extracellular vesicles can interact with endothelial cells and modulate their cellular functions. Thrombospondin-1 and its receptor CD47 are expressed on exosomes/ectosomes derived from T cells, and these extracellular vesicles are internalized and modulate signaling in both T cells and endothelial cells. Extracellular vesicles released from cells expressing or lacking CD47 differentially regulate activation of T cells induced by engaging the T cell receptor. Similarly, T cell-derived extracellular vesicles modulate endothelial cell responses to vascular endothelial growth factor and tube formation in a CD47-dependent manner. Uptake of T cell derived extracellular vesicles by recipient endothelial cells globally alters gene expression in a CD47-dependent manner. CD47 also regulates the mRNA content of extracellular vesicles in a manner consistent with some of the resulting alterations in target endothelial cell gene expression. Therefore, the thrombospondin-1 receptor CD47 directly or indirectly regulates intercellular communication mediated by the transfer of extracellular vesicles between vascular cells.
Project description:Exosomes are small membrane bound cell-derived vesicles that are present in biological fluids include blood and cell culture medium. Exosomes contain various functional proteins, mRNAs and microRNAs (miRNAs). We used miRNA microarrays to detail the miRNA content in the GW627368-induced and PGE2-induced exosomes from non-adherent mammary epithelial cells (NAMECs).
Project description:Exosomes are small membrane bound cell-derived vesicles that are present in biological fluids include blood and cell culture medium. Exosomes contain various functional proteins, mRNAs and microRNAs (miRNAs). We used miRNA microarrays to detail the miRNA content in plasma exosomes of mice bearing A549Ago2-KO/HA-Ago2Wt and A549Ago2-KO/HA-Ago2Δ (Dm) tumors.
Project description:Elevated expression of CD47 in some cancers is associated with poor survival, related to its function as an innate immune checkpoint when expressed on tumors cells. In contrast, elevated CD47 ex-pression in cutaneous melanomas is associated with improved survival. Previous studies impli-cated protective functions of CD47 expressed by immune cells in the melanoma tumor microen-vironment. RNA sequencing analysis of responses induced by CD3 and CD28 engagement on wild type and CD47-deficient Jurkat T lymphoblast cells identified additional regulators of T cell func-tion that were also CD47-dependent in mouse CD8 T cells. MYCN mRNA expression was up-regulated in CD47-deficient cells but down-regulated in CD47-deficient cells following activa-tion. CD47 also regulated alternative splicing that produces two N-MYC isoforms. The CD47 ligand thrombospondin-1 inhibited expression of these MYCN mRNA isoforms as well as induction of the oncogenic decoy MYCN opposite strand (MYCNOS) noncoding RNA during T cell activation. Analysis of mRNA expression data for melanomas in The Cancer Genome Atlas identified signif-icant coexpression of MYCN with CD47 and known regulators of CD8 T cell function. Throm-bospondin-1 inhibited the induction of TIGIT, CD40LG and MCL1 mRNAs following T cell acti-vation in vitro. Increased mRNA expression of these T cell transcripts and of MYCN in melanomas was associated with improved overall survival.
Project description:Exosomes are small extracellular nano-vesicles of endocytic origin that mediate different signals between cells, by surface interactions and by shuttling of functional RNA from one cell to another. In this study, we show that exosomes, produced by mouse mast cells exposed to oxidative stress, change their mRNA content and also that these exosomes can influence the response of other cells to oxidative stress by providing recipient cells with a resistance against oxidative stress. Finally, we also show that UV-light affect the biological functions associated with exosomes released under oxidative stress. These results argue that exosomal shuttle of RNA is involved in cell-to-cell communication, by influencing the response of recipient cells to an external stimulus. We used microarrays to detail to examine the gene expression underlying the effect of H2O2 on the exosomal RNA and how exosomes isolated from oxidative stress influence the growth of recipient cells during oxidative stress. The mRNA content of the exosomes from normal conditions (MC9 Exo_norm) were compared to that from exosomes from H2O2 conditions (MC9 Exo_H2O2). The mRNA content of recipient cells MC9 were identified before (MC9 cells_norm) and after (MC9 cells_H2O2) addition of exosomes that were isolated from normal and H2O2 conditions.
Project description:Extracellular vesicles (EVs) mediate cell-cell communication including the intercellular transfer of miRNAs, which alter gene expression in target cells. Previously, we have shown that CD47 is also present on EVs and alters their effects on target cells, suggesting that specific surface markers define functionally distinct EVs. This hypothesis will be addressed by comparing total cellular versus EVs miRNA contents between Jurkat and JinB8 (CD47 deficient) T cells. The Jurkat T cell line (E6.1) was purchased from ATCC. The cells were maintained using RPMI 1640 containing 10% FBS, glutamine, penicillin, and streptomycin (Gibco). The Jurkat and JinB8 T cells used for experiments were maintained less than 6 weeks in culture. The cultured Jurkat were washed with IXPBs followed by a second wash with HITES medium (DMEM/F12 supplemented with, bovine serum albumin, hydrocortisone, insulin, transferrin, and trace elements). EVs were isolated from Jurkat T cells were cultured overnight using HITES medium. The conditioned media from Jurkat T cells were collected, and cell debris were removed using centrifugation at 300 g for 5 minutes and 2500 g for 10 minutes respectively. Centrifuged media were concentrated using Ultra-4 centrifugal filters to about 1 ml volume and further centrifuged at 10,000 g for 10 minutes. EVs were isolated using an Exo-Quick kit (SBI). miRNaseazy (Qiagen) kits were used to extract total RNA from Jurkat and JinB8 T cells and their EVs according to the manufacturer's instructions.