Care and handling of animals and all experimental procedures used here were approved under protocol LP-012 and in compliance with standards established by the Animal Care and Use Committee of the National Cancer Institute

Care and handling of animals and all experimental procedures used here were approved under protocol LP-012 and in compliance with standards established by the Animal Care and Use Committee of the National Cancer Institute. Cells Mouse lung endothelial cells were isolated and their purity verified as described previously50. 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. CD47 is a signaling receptor for the secreted matricellular protein thrombospondin-1 and the counter-receptor for signal-regulatory protein- (SIRP), which on phagocytic cells recognizes CD47 engagement as a marker of self1,2,3. Mice lacking CD47 or thrombospondin-1 are profoundly resistant to tissue stress associated with ischemia, ischemia/reperfusion, and high dose irradiation2,4,5,6,7. The survival advantage of ischemic CD47- and thrombospondin-1-null tissues is mediated in part by increased nitric oxide/cGMP signaling2. Radioresistance associated with CD47 blockade is cell autonomous and independent of NO signaling8, indicating that additional pro-survival signaling pathways are controlled by CD47. Engaging CD47 in some cell types triggers programmed cell death3,9. BCL2/adenovirus E1B 19?kDa protein-interacting protein 3 (BNIP3) is a pro-apoptotic BH3 domain protein that interacts with the cytoplasmic tail of CD47 and is implicated in CD47-dependent cell death10. Furthermore, CD47 ligation alters localization of the dynamin-related protein Drp1, which controls mitochondria-dependent death pathways9, and some tissues TUG-770 in CD47-null and thrombospondin-1-null mice show increased mitochondrial numbers and function11. Mitochondrial-dependent cell death pathways involving Bcl-2 are limited by the autophagy regulator beclin-112. We TUG-770 recently found that CD47 signaling limits the induction of beclin-1 and other autophagy-related proteins in irradiated cells, and blocking CD47 in vitro and in vivo thereby increases activation of a protective autophagy response13,14. This autophagy response is necessary for the radioprotective effect of CD47 blockade. In contrast to the above noted survival advantages of decreased CD47 expression, elevated expression of CD47 confers an indirect survival advantage in vivo. CD47 engages SIRP on macrophages and prevents phagocytic clearance1,15. Similarly, elevated expression of CD47 on several types of cancer cells has been shown to inhibit their killing by macrophages or NK cells16,17,18. Conversely, CD47 antibodies that block SIRP binding enhance macrophage-dependent clearance of tumors17,19,20,21, although others have shown that such clearance can occur independent of inhibitory SIRP signaling22,23,24. Taken together, these studies indicate two opposing roles for CD47 in cell survival. The cell autonomous advantages of decreased CD47 expression, leading to less inhibitory CD47 signaling, must be balanced against the need to maintain sufficient CD47 levels to prevent phagocytic clearance in vivo. Hematopoietic stem cells exhibit elevated CD47 expression, and high CD47 expression in the stem cell niche was proposed to be important to protect stem cells from innate immune surveillance25. In contrast to this protective function of CD47 in stem cells, we now report that loss of CD47 elevates expression of the stem cell transcription factors Sox2, Klf4, Oct4, and c-Myc in primary murine endothelial cells. Consequently, these cells exhibit increased asymmetric cell division and spontaneously and efficiently form clusters that resemble Cdc14A2 embryoid bodies (EBs) in serum-free media without requiring feeder cells. These EB-like clusters can readily differentiate into various lineages. c-Myc is a global regulator of gene expression in differentiated and stem cells26 and plays a major role in this inhibitory function of CD47. Re-expression of CD47 in null cells down-regulates c-Myc expression and inhibits cell growth, whereas dysregulation of the gene, such as commonly occurs in cancer, enables cells to tolerate high CD47 expression. Results Loss of CD47 allows self-renewal and increases c-Myc expression Primary cells isolated from CD47-null mice exhibit a remarkable advantage in adapting to the stress of tissue culture. Lung endothelial cells isolated from WT C57Bl/6 mice had limited survival and proliferative capacities in primary culture as assessed by reduction TUG-770 of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and.

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