CD47 was first identified in 1987 as an antigen that is missing in red blood cells of patients with Rhesus (Rh)-null hemolytic anemia (see the following reviews for CD47 source references unless otherwise cited: Rogers et al. 2012; Soto-Pantoja et al. 2015; Soto-Pantoja et al. 2013). Loss of CD47 is not the primary cause of this disease, but CD47 closely associates with the Rh complex on red blood cells. Independently, the same protein was identified as an antigen, OA3, that is overexpressed in ovarian carcinoma and as a protein that copurified with certain integrins and named integrin-associated protein (IAP). IAP and OA3 were shown in 1994 to be identical to CD47. CD47 is a type I integral membrane protein with an extracellular immunoglobulin variable (IgV)-like domain, five membrane-spanning segments, and a short alternatively spliced carboxy-terminal cytoplasmic tail. The four splice variants have unique cell and tissue distribution patterns. CD47 is widely expressed in higher vertebrates. In addition to its lateral association with certain integrins and the Rh complex, three extracellular ligands have been identified for CD47: thrombospondin-1 (TSP1), SIRPα (also known as Src homology 2 domain containing protein tyrosine phosphatase substrate-1 or SHPS1), and SIRPγ. TSP1 is a secreted protein and is the most studied ligand for regulating signaling through CD47. SIRPs are integral membrane proteins with important signaling functions, and CD47 serves as a counterreceptor for SIRPα. Most studies of SIRP-CD47 interactions regard CD47 as the ligand that regulates signal transduction through SIRPα (Chao et al. 2012; Barclay and van den Berg 2014), which will not be discussed here. However, there is some evidence that this signaling is bidirectional (Sarfati et al. 2008), and more research is needed to clarify the role of SIRPs as ligands that regulate CD47 signaling.
Early studies of CD47 function as a signaling receptor focused on its role in integrin activation, and peptides derived from TSP1 that bind to CD47 were shown to enhance the activation of specific integrins. Ligation of CD47 by these peptides in different cell types also stimulated calcium influx and changes in cAMP levels, MAP kinase activities, and Giα activation. However, evidence for CD47 signaling that relies exclusively on these peptides must be viewed with skepticism based on compelling evidence that the same peptides can induce signaling independent of CD47 (Soto-Pantoja et al. 2015).
The central function of CD47 in cardiovascular physiology was first revealed by an enhanced angiogenic response to nitric oxide (NO) in muscle explants from Thbs1- and CD47-null mice. TSP1 binding to CD47 inhibited NO-mediated activation of soluble guanylate cyclase (sGC) in endothelial cells, and this inhibitory effect was lost in endothelial cells from CD47-null mice. Subsequent studies extended this inhibition of NO signaling to other vascular cells, including smooth muscle cells, platelets, and T lymphocytes. Functional studies showed that this inhibitory signaling by TSP1 through CD47 acutely controls vascular tone, blood pressure, and platelet hemostasis (Rogers et al. 2012). Subsequent biochemical studies expanded the intracellular targets of this inhibitory signaling to include cGMP-dependent protein kinase (cGK), endothelial nitric oxide synthase (eNOS, NOS3), and vascular endothelial growth factor receptor-2 (VEGFR2) (Rogers et al. 2012). Thus, CD47 signaling is now recognized as a highly redundant inhibitor of the canonical NO/cGMP signaling cascade in vascular cells.
A second major function of CD47 signaling is to control cell survival. Early studies showed that some CD47 antibodies can induce apoptosis of T cells. Studies in mice lacking CD47 have extended this more broadly to survival of ischemic stress, ischemia reperfusion, global hypoxia, tissue and organ transplantation, and radiation injury (Soto-Pantoja et al. 2015; Rogers et al. 2016). Mechanistic studies show some involvement of the NO/cGMP cascade and regulation of SIRPα-mediated phagocytosis, but additional pathways have been identified through which CD47 signaling controls mitochondrial-dependent cell death and a protective autophagy response (Soto-Pantoja et al. 2015).
Proximal Targets of CD47
CD14 is another lateral interaction partner that controls CD47 signaling in macrophages (Stein et al. 2016) (Fig. 1). CD14 constitutively associates with CD47, but TSP1 binding to CD47 dissociates CD14 and inhibits its function as a coreceptor for lipopolysaccharide (LPS) to mediate proinflammatory signaling leading to synthesis and release of active IL-1β.
Several cytoplasmic binding partners of CD47 have been identified, including some heterotrimeric G proteins. Ligation of CD47 leads to dissociation of Giα from Gβγ. The ability of cyclodextrin to dissociate Giα from CD47 suggests that this interaction is not direct and may involve membrane cholesterol. Yeast two-hybrid studies revealed that protein linking IAP and cytoskeleton-1 (PLIC-1, ubiquilin-1) and the related PLIC-2 (ubiquilin-2) are proximal cytoplasmic ligands of CD47. These ubiquilins play diverse roles in the ubiquitin–proteasome system and autophagy (Zhang et al. 2014), but their specific role in CD47 signaling remains unclear. Another yeast 2-hybrid screen identified Bcl2 homology 3-only protein 19 kDa interacting protein-3 (BNIP3) as an interaction partner of CD47.
TSP1 binding to CD47 potently limits sGC activation by NO in endothelial and vascular smooth muscle cells and platelets (Rogers et al. 2012). TSP1 also potently inhibits sGC activation by heme-dependent and nonheme-dependent chemical activators such as YC-1 and Riocuguat (BAY 63-2521) in vascular cells (Miller et al. 2010). Physiological circulating concentrations of TSP1 in blood (0.1–0.2 nM) are sufficient to partially inhibit sGC activation. This is consistent with the observation that endothelial and vascular smooth muscle cells and platelets from Thbs1- and CD47-null mice have elevated basal levels of cGMP. In wounds and some chronic disease states, much greater levels of TSP1 can be demonstrated in tissues and blood. These findings suggest that TSP1 functions in both health and disease to inhibit NO-stimulated activation of sGC. At higher concentrations (>10 nM), TSP1 also inhibits sGC activation via engaging the cell receptor CD36, which is restricted to microvasculature, but this inhibitory activity requires the presence of CD47.
TSP1 limits activation of cGK in platelets stimulated by NO or by cell permeable analogs of cGMP (Rogers et al. 2012) (Fig. 2). Furthermore, assay of cGK activity using a defined peptide substrate in lysates of platelets pretreated with TSP1 showed inhibition of in vitro activation of the enzyme by cGMP. Therefore, cGK is a second direct target of CD47 signaling (Fig. 2).
eNOS is the major NO synthase expressed in vascular endothelium. eNOS is activated by hormonal or mechanical stimulation of the endothelium through modulation of intracellular calcium and phosphorylation of eNOS. TSP1 inhibits eNOS activation in endothelial cells via CD47, thereby limiting endothelial-dependent arterial dilation (Rogers et al. 2012). CD47-null mice are hypotensive at rest compared to wild-type controls, demonstrating a role for the cell surface receptor as a mediator of arterial tone and vascular resistance. In vivo circulating TSP1 was found to limit endothelial activation and associated decreases in blood pressure (Rogers et al. 2012). Thus, physiologic levels of circulating TSP1 function as a hypertensive by limiting eNOS activation and endogenous NO production, and in this capacity TSP1 functions to support blood pressure. Targeting the TSP1-CD47 ligand receptor interaction can acutely lower blood pressure, suggesting possible therapeutic opportunities in the treatment of systemic hypertension.
Phosphorylation of eNOS activates the enzyme and can be stimulated in endothelial cells by VEGF binding to its receptor VEGFR2 (Fig. 2). CD47 interacts directly with VEGFR2 in endothelial cells and T cells (Soto-Pantoja et al. 2015). Ligation of CD47 in the presence of VEGF dissociates CD47 from VEGFR2 and prevents autophosphorylation of VEGFR2 at Tyr1175. This is a necessary phosphorylation for downstream signaling. Therefore, CD47 ligation by TSP1 globally inhibits signaling downstream of VEGFR2, which includes Src kinase and phospholipase Cγ pathways important for cell migration and proliferation in addition to blocking Akt and subsequent eNOS activation (Fig. 2).
Cell-Survival Signaling and Autophagy
Cell death involving CD47 is not exclusive to T cells. In vitro treatment with a specific immobilized CD47 antibody or TSP1 induces apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cell clones from patients. As observed in T cells, CLL cell death occurred without nuclear features such as chromatin condensation and DNA fragmentation. However, cytoplasmic changes, such as cell shrinkage, and decrease in mitochondrial membrane potential were observed (Sarfati et al. 2008). Moreover, electron microscopy revealed swelling of the mitochondria, indicating an increase in permeability of the mitochondrial membrane. This indicates that CD47 may regulate mitochondrial function to control cell survival. In activated T cells, treatment with immobilized CD47 antibody B6H12 disrupts mitochondrial transmembrane potential, followed by the release of reactive oxygen species (Roue et al. 2003). This dysfunction in the mitochondria is not accompanied by the release of cytochrome C or AIF, indicating an alternate mechanism for programmed cell death (Roue et al. 2003).
One possible mediator of CD47-dependent cell death is BNIP3, a member of the Bcl2 interacting proteins that translocates to the mitochondria to induce apoptosis (Fig. 4). BNIP3 contains a BH3 domain and a transmembrane domain that are associated with proapoptotic functions. BNIP3 interacts with the transmembrane domains of CD47. Antisense suppression of BNIP3 inhibited CD47-mediated induction of apoptosis, indicating that binding of antibody or peptides mimicking TSP1 induces BNIP3 to translocate from the plasma membrane to the mitochondria to execute cell death. This regulation of mitochondrial function by CD47 implicates other forms of cell death.
CD47 ligation also regulates dynamin-related protein 1 (Drp1), a major regulator of type III programmed cell death (Bras et al. 2007). CD47 ligation induces Drp1 translocation from the cytosol to mitochondria, a process controlled by chymotrypsin-like serine proteases. Once in mitochondria, Drp1 blocks the mitochondrial electron transport chain, which dissipates the mitochondrial transmembrane potential, increases reactive oxygen species generation, and produces a drop in ATP levels (Bras et al. 2007). Knockdown of Drp1 using siRNA prevented the loss of mitochondrial function after ligation of CD47 by the immobilized antibody B6H12, suggesting that Drp1 is essential for the CD47 regulation of mitochondrial function and induction of type III–programmed cell death.
CD47 also controls cell survival independent of controlling programmed cell death by allowing senescent or damaged cells to escape phagocytosis (Barclay and van den Berg 2014). CD47 is upregulated in a number of cancers, including ovarian and bladder carcinomas and myeloid leukemia and in migrating hematopoietic progenitors. Increased CD47 expression correlates with an ability to evade phagocytosis by macrophages and cytolysis by NK cells (Chao et al. 2012). Conversely, CD47 expression cell-autonomously limits survival of many cell types under conditions of stress (Rogers et al. 2012). In fixed ischemic stress generated by surgically creating myocutaneous flaps, tissue blood flow and perfusion were dramatically increased in Thbs1- and CD47-null animals. Similar observations have been made for other fixed ischemic insults or ischemia/reperfusion injuries in mice, rats, and pigs. In a model of focal cerebral ischemia, CD47 null mice show reduced brain damage after ischemic insult. The activity of CD47 to limit tissue survival under ischemic stress is based on its inhibition of NO/cGMP-mediated maintenance of tissue perfusion and vascular remodeling. Treatment with antibodies to CD47 or antisense morpholino oligonucleotides that suppress CD47 mRNA translation increases recovery from ischemic insults in several animal models. Together, these data suggest that therapeutic blockade of CD47 could be clinically useful to restore blood flow and improve tissue survival after ischemic and ischemic/reperfusion injuries.
TSP1/CD47 signaling further regulates inflammatory responses and cell survival through activation of the inflammasome pathway (Stein et al. 2016). Binding of TSP1 to CD47 inhibits the transcriptional upregulation by LPS of pro-IL-1β, NLRP3, and caspase-1 mRNAs but enhances its subsequent activation. CD47 promotes LPS-dependent transcription of IL-1β. Ligation of CD47 by TSP1 inhibits the former LPS (signal one)-dependent inflammasome induction by disrupting the interaction between CD14 and CD47. Regulation of IL-1β release by CD47 may in turn regulate cell survival during inflammation.
The cytoprotective effects of decreased CD47 expression extend to ionizing radiation (IR) injuries. IR acutely damages cellular DNA and other cellular macromolecules, eliciting stress responses that ultimately lead to cell death. CD47- and Thbs1-null mice show enhanced skin, muscle, and bone marrow preservation after radiation injury. Vascular endothelial cells cultured from the null mice showed enhanced cell survival and proliferative capacity after IR injury, demonstrating that this acute radioprotection of vascular cells is cell-autonomous. Antisense suppression of CD47 protected human endothelial cells in vitro and protected soft tissue, bone marrow, and tumor-associated leukocytes in irradiated hindlimbs of mice (Maxhimer et al. 2009) and mice subjected to lethal total body irradiation. Although NO is a known radioprotectant, enhanced NO signaling is not sufficient to account for radioprotection by CD47 blockade. Treatment with DETA/NO or a cell permeable cGMP analog did not result in a significant survival advantage to irradiated endothelial cells (Maxhimer et al. 2009). Conversely, inhibition of endogenous NO production using L-NAME did not prevent the protective effects of CD47 morpholino (Maxhimer et al. 2009). Therefore, the radioprotection observed by CD47 targeting occurs in a largely NO-independent manner.
The radioprotection achieved by CD47 blockade is mediated by the autophagy pathway (Soto-Pantoja et al. 2015) (Fig. 4). Cells lacking CD47 exhibit enhanced basal autophagy and stronger induction of autophagic flux after exposure to IR based on increased LC3 puncta formation and p62/sequestrome-1 degradation. CD47 signaling limits mRNA and protein expression levels of beclin-1, ATG5, and ATG7, and siRNA knockdown of ATG5 or ATG7 blocked this CD47 response. Autophagy alters a number of cellular metabolic pathways, and correspondingly CD47-deficient cells exposed to IR exhibit global preservation of metabolic pathways involved in energy metabolism, DNA repair, and oxidative stress responses (Miller et al. 2015).
In contrast to its radioprotective activity in nonmalignant tissues, the suppression of CD47 sensitized syngeneic tumors in mice to radiation treatment. CD47 morpholino treatment followed by IR dramatically delayed tumor regrowth relative to IR alone (Maxhimer et al. 2009). In vitro, CD47 blockade conferred minimal radioprotection to tumor cells, suggesting that suppression of CD47 enhances tumor growth delay after irradiation through enhancing host antitumor immunity (Maxhimer et al. 2009). Subsequent studies demonstrated that CD8 T cells are required for the synergism between CD47 blockade and tumor irradiation (Soto-Pantoja et al. 2014).
T Cell Signaling, Differentiation, and Tumor Cell Killing
Inhibitory CD47 signaling in CD8+ T cells inhibits their antigen-dependent cytotoxic activity (Soto-Pantoja et al. 2014). Suppressing CD47 expression in either CD8+ T cells or target tumor cells increased target cell killing. Genetic deletion of CD47 in host T cells was sufficient to increase CD8+ T cell-dependent ablation of syngeneic tumors in mice. Therapeutic blockade or loss of CD47 elevated expression of granzyme B in T cells and tumor cells, identifying this cytolytic enzyme as a target of CD47 signaling.
Other Second Messengers and Transcriptional Regulation
Studies of CD47 signaling in stem cells have identified several transcription factors as targets of CD47 signaling (Kaur and Roberts 2016). TSP1 inhibited the angiogenic potential of endothelial precursor cells by a CD47-dependent mechanism. Similarly, CD47- and Thbs1-null mouse lung endothelial cells exhibit increased proliferative capacity, which is due to increased expression of the four Yamanaka transcription factors cMyc, Sox2, Oct4, and Klf4 (Soto-Pantoja et al. 2015). The same transcription factors were elevated in the kidney of CD47-null mice after ischemia reperfusion injury and in isolated renal tubular epithelial cells (Rogers et al. 2016). TSP1 and a TSP1-derived CD47 binding peptide downregulated expression of cMyc only in murine or human cells that express CD47. In contrast, cancer cells with dysregulated cMyc such as Burkitt’s lymphoma were insensitive to TSP1 or overexpression of CD47. A subsequent study in human breast cancer stem cells as well as studies in liver and pancreatic cancer stem cells indicated that CD47 signaling supports rather than suppresses cancer stem cells (Kaur and Roberts 2016). In breast cancer stem cells, a CD47 blocking antibody decreased expression of the stem cell transcription factor Klf4. Therefore, CD47 signaling to control stem cell self-renewal involves divergent signaling pathways in normal versus transformed cells. This provides an explanation for the apparently conflicting observations that loss of CD47 enhances stem cells in healthy tissues, whereas cancer stem cells typically express elevated levels of CD47.
CD47 is a ubiquitously expressed transmembrane receptor for two members of the SIRP/SHPS family and for the secreted protein TSP1. Signaling between CD47 and SIRPα can be bidirectional, but signaling through CD47 in response to SIRPα ligation remains poorly characterized. TSP1 binding to CD47 alters its lateral interactions with several integrins, VEGFR2, Fas, CD14, and SIRPα. This results in altered signaling through each of these receptors and CD47 to modulate cell metabolism, adhesion, survival, self-renewal, contractility, proliferation, and motility. Downstream signaling through CD47 controls levels of the second messengers, calcium, cAMP, and cGMP, in a cell-specific manner. In vascular cells, NO is a major physiological target of CD47 signaling. CD47 signaling redundantly controls both synthesis and effector pathways downstream of NO, enabling it to control both intracellular and intercellular signaling between vascular cells. This signaling has physiological roles in angiogenesis, cardiovascular dynamics, and hemostasis. Antagonism of NO signaling by CD47 also limits tissue responses to ischemic stress and systemic hypoxia. In contrast, regulation of cell survival after irradiation requires autophagy and involves global alterations in cell metabolism that are controlled by CD47 signaling. CD47 signaling controls stem cell self-renewal by regulating the expression of cMyc and other stem cell transcription factors.
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