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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIASC009172-25
Application #
8763691
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
25
Fiscal Year
2013
Total Cost
$945,821
Indirect Cost
Name
National Cancer Institute Division of Clinical Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Rogers, Natasha M; Seeger, Franziska; Garcin, Elsa D et al. (2014) Regulation of soluble guanylate cyclase by matricellular thrombospondins: implications for blood flow. Front Physiol 5:134
Soto-Pantoja, David R; Shih, Hubert B; Maxhimer, Justin B et al. (2014) Thrombospondin-1 and CD47 signaling regulate healing of thermal injury in mice. Matrix Biol 37:25-34
Kaur, Sukhbir; Singh, Satya P; Elkahloun, Abdel G et al. (2014) CD47-dependent immunomodulatory and angiogenic activities of extracellular vesicles produced by T cells. Matrix Biol 37:49-59
Kaur, Sukhbir; Chang, Tiffany; Singh, Satya P et al. (2014) CD47 signaling regulates the immunosuppressive activity of VEGF in T cells. J Immunol 193:3914-24
Rogers, Natasha M; Roberts, David D; Isenberg, Jeffrey S (2013) Age-associated induction of cell membrane CD47 limits basal and temperature-induced changes in cutaneous blood flow. Ann Surg 258:184-91
Soto-Pantoja, David R; Ridnour, Lisa A; Wink, David A et al. (2013) Blockade of CD47 increases survival of mice exposed to lethal total body irradiation. Sci Rep 3:1038
Isenberg, Jeff S; Qin, Yan; Maxhimer, Justin B et al. (2009) Thrombospondin-1 and CD47 regulate blood pressure and cardiac responses to vasoactive stress. Matrix Biol 28:110-9
Isenberg, Jeff S; Martin-Manso, Gema; Maxhimer, Justin B et al. (2009) Regulation of nitric oxide signalling by thrombospondin 1: implications for anti-angiogenic therapies. Nat Rev Cancer 9:182-94
Maxhimer, Justin B; Soto-Pantoja, David R; Ridnour, Lisa A et al. (2009) Radioprotection in normal tissue and delayed tumor growth by blockade of CD47 signaling. Sci Transl Med 1:3ra7
Isenberg, Jeff S; Annis, Douglas S; Pendrak, Michael L et al. (2009) Differential interactions of thrombospondin-1, -2, and -4 with CD47 and effects on cGMP signaling and ischemic injury responses. J Biol Chem 284:1116-25

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