CD47 signaling pathways controlling cellular differentiation and responses to stress

Abstract

CD47 is a widely expressed integral membrane protein that serves as the counter-receptor for the inhibitory phagocyte receptor signal-regulatory protein-α (SIRPα) and as a signaling receptor for the secreted matricellular protein thrombospondin-1. Recent studies employing mice and somatic cells lacking CD47 have revealed important pathophysiological functions of CD47 in cardiovascular homeostasis, immune regulation, resistance of cells and tissues to stress and chronic diseases of aging including cancer. With the emergence of experimental therapeutics targeting CD47, a more thorough understanding of CD47 signal transduction is essential. CD47 lacks a substantial cytoplasmic signaling domain, but several cytoplasmic binding partners have been identified, and lateral interactions of CD47 with other membrane receptors play important roles in mediating signaling resulting from the binding of thrombospondin-1. This review addresses recent advances in identifying the lateral binding partners, signal transduction pathways and downstream transcription networks regulated through CD47 in specific cell lineages. Major pathways regulated by CD47 signaling include calcium homeostasis, cyclic nucleotide signaling, nitric oxide and hydrogen sulfide biosynthesis and signaling and stem cell transcription factors. These pathways and other undefined proximal mediators of CD47 signaling regulate cell death and protective autophagy responses, mitochondrial biogenesis, cell adhesion and motility and stem cell self-renewal. Although thrombospondin-1 is the best characterized agonist of CD47, the potential roles of other members of the thrombospondin family, SIRPα and SIRPγ binding and homotypic CD47 interactions as agonists or antagonists of signaling through CD47 should also be considered.

Keywords: Autophagy; Myc; hydrogen sulfide; immune regulation; integrin signaling; nitric oxide; radioresistance; stem cells.

Fig. 1

Topological arrangements of CD47 and its known extracellular ligands. (A) Thrombospondin-1 (TSP1) is a secreted protein that binds to a proteoglycan isoform of CD47 on the plasma membrane. Mutagenesis studies established that heparan sulfate modification of CD47 is required for high affinity interaction of TSP1 with CD47. (B) Two members of the signal recognition protein family (SIRPα and SIRPγ) bind to CD47 in a species-specific manner. These serve as counter-receptors for CD47 during cell-cell interactions, although evidence in smooth muscle cells suggests SIRPα can also interact in-cis with CD47 (Maile and Clemmons, 2003). Proteolytic cleavage of the extracellular domain of SIRPα can also generate a soluble ligand for CD47 (Umemori and Sanes, 2008). (C). Homotypic binding of CD47 to CD47 or binding to an unidentified trypsin-sensitive counter-receptor (X) can mediate cell-cell interactions (Rebres et al., 2005). The IgV domain of CD47 can be shed under some conditions (Maile et al., 2008b, Kaur et al., 2011), but the ability of this domain to engage in homotypic binding as a soluble CD47 ligand is not known.

Fig. 2

Evidence for the relationship between TSP1 and SIRPα binding sites on CD47. (A) Data from Isenberg et al (Isenberg et al., 2009a) demonstrates that TSP1 and the function-blocking CD47 antibody B6H12 inhibit binding of the radiolabeled extracellular domain of SIRPα to cells expressing CD47. Lack of binding to an isogenic cell line lacking CD47 confirms specificity of SIRPα binding. These competition data could indicate that TSP1 and SIRPα bind to overlapping sites on CD47 or that binding of these proteins to distinct sites on CD47 results in steric or allosteric inhibition of SIRPα binding by TSP1. (B) A space-filling projection of the crystal structure of the extracellular IgV domain of human CD47 (tan backbone with colored side chains) complexed with the terminal Ig domain of SIRPα (yellow). Mutagenesis established that post-translational modification of Ser⁶⁴ on CD47 (yellow) is required for TSP1 signaling. This suggests that the TSP1 binding site is distinct from the SIRPα binding site on CD47.

Fig. 3

Supramolecular complexes containing CD47. (A) On a majority of cell types CD47 laterally associates with specific integrins. These integrins in turn may associate with other membrane proteins, potentially including CD36 in neuronal cells (Bamberger et al., 2003). However, readers are cautioned that the proposed CD36-α6β1-CD47 complex has not been directly demonstrated. PLIC1/ubiquilin-1 directly binds to the C-terminal cytoplasmic tail of CD47 and can recruit heterotrimeric G-proteins to CD47. (B) In T cells CD47 can laterally associate with Fas receptor. Yeast two-hybrid screening identified BNIP3 as another direct binding partner for the C-terminal tail of CD47. (C) In innate immune cells FRET studies identified lateral association of CD47 with the LPS co-receptor CD14. CD14 is also a component of the TLR4 signaling complex, but it is unclear whether CD447 can bind to CD14 while it is in the TLR complex. (D) In endothelial cells CD47 closely associates with VEGFR2. This complex also contains Src kinases, which associate with the cytoplasmic tail of VEGFR2 via TSAd (Shibuya, 2006). (E) Human erythrocytes lack integrins, and CD47 is found in a multiprotein complex that accounts for Rh-antigen activity. This complex also contains Band 3 and is anchored to the red cell cytoskeleton via ankyrin. Band 4.2 is required for maintaining CD47 in this complex. (F) Lateral oligomerization of CD47 has been reported in association with lipid rafts. Heterotrimeric G protein and the Src kinases Lyn and Fyn are enriched in this complex.

Fig. 4

Functional crosstalk between CD36 and CD47 signaling. In vascular cells ligation of CD36 by amyloid-β or a peptide derived from the type 1 repeats of TSP1 inhibits uptake of free fatty acids via CD36, which regulates eNOS, and inhibits downstream NO/cGMP signaling. The latter pathway requires CD47, but the mechanism remains unclear. One possibility is the lateral association of CD36 with an integrin that also associates with CD47.

Fig. 5

Extracellular signaling by CD47. CD47 is released by cells in extracellular vesicles known as exosomes. Exosomes are taken up by endothelial cells and T cells and enable transfer of CD47 and the RNA cargo of exosomes to target cells that can alter gene expression and functional responses in the recipient cells in a CD47-dependent manner (Kaur et al., 2014b).

Fig. 6

CD47 regulation of nitric oxide signaling in vascular cells. TSP1 signaling through CD47 redundantly inhibits NO signaling that the level of eNOS, sGC, and cGK. This blocks signaling initiated by upstream calcium- and phosphorylation-mediated activation of eNOS as well as activation by exogenous NO, sGC activating drugs or phosphodiesterase inhibitors, or the direct cGK activator 8-bromo-cGMP. By dissociating the lateral interaction of CD47 with VEGFR2, TSP1 further inhibits upstream signals that activate eNOS and globally inhibits NO-independent branches of the VEGFR2 signaling cascade.

Fig. 7

CD47 regulation of autophagy. In the presence of cellular stress, blockade of CD47 activates gene expression of beclin-1 and autophagy related proteins (ATG) 5 and 7 that limit BCL-2 mediated pathways to induce cell death. The increase in beclin-1 ATG5 and ATG7 leads to increased autophagic flux by increasing expression of LC3 to form the autophagosome membrane and stimulates p62, which targets unwanted cargo to be degraded in the autophagosome. The radioprotective effect of CD47 is reversed by the administration of 3-methyladenine (3-MA), which inhibits early activation of autophagy, or the lysosomotropic agent hydroxychloroquine (HCQ), which inhibits autophagy at late stages of the pathway.

Fig. 8

CD47 signaling limits stem cell self-renewal. Withdrawal of serum from differentiated cd47-null cells induces their spontaneous reprogramming to multipotent stem cells that can differentiate along all embryonic lineages. Suppression of CD47 in wild type cells using a CD47 morpholino increases expression of stem cell transcription factors and increased asymmetric cell division, whereas treatment with TSP1 or a CD47-binding peptide derived from TSP1 suppresses the stem cell phenotype.

Fig. 9

CD47 regulation of T cell activation and H₂S signaling. T cells are activated by T cell receptor (TCR) signaling in response to recognition of antigens presented in the context of MHC or experimentally using an anti-CD3 antibody. TSP1 signaling through CD47 inhibits TCR signaling via several mechanisms including inhibiting induction of the H₂S biosynthetic enzymes cystathionine β–synthase (CBS) and cystathionine γ–lyase (CSE). H₂S produced by CBS and CSE increases actin polymerization and reorients the microtubule organizing center (MTOC) to enhance immunological synapse formation with antigen-presenting cells.