CD47: A Master Regulator of Stemness
Embryoid bodies spontaneously formed by CD47-null cells highly express the stem cell transcription factor Sox2, which is indicated by green staining. Cell nuclei in this image are stained blue.
Identifying the pathways cells use to regulate proliferation, differentiation, and survival are essential for designing new treatments to stimulate organ and tissue repair following injury and for diseases as diverse as cancer and diabetes. The thrombospondin-1 receptor CD47 seems to limit cell survival and regeneration after stress. At the same time, CD47 levels are increased on the surface of cancer cells, which show enhanced proliferation and survival. To understand this apparent paradox, David Roberts, Ph.D., in CCR’s Laboratory of Pathology and his colleagues decided to investigate CD47’s mechanism of action using CD47- and thrombospondin-1-null mice.
The researchers began their studies by isolating lung endothelial cells from wild type, CD47-null, and thrombospondin-1-null mice. Unlike wild type cells that showed limited proliferation and survival in culture, the cells lacking CD47 or thrombospondin-1 readily produced continuously proliferating cell lines that retained a differentiated morphology and endothelial cell markers. Of the genes associated with escape from senescence or induction of a stem cell-like phenotype, only c-Myc mRNA and protein levels were significantly elevated. The investigators then looked at the expression of additional transcription factors associated with stemness. They found elevated levels of the stem cell transcription factors Sox2, Klf4, and Oct4 as well as other stem cell markers, suggesting that CD47-null cells were enriched for stem cells.
Consistent with this idea, the scientists found that cells lacking CD47 divided asymmetrically, with one cell retaining both original strands of DNA, more often than wild type cells. Additionally, growing the CD47-null cells in serum-free medium induced the formation of cell aggregates resembling embryoid bodies (EBs), a hallmark of induced pluripotent stem cells. Treating the CD47-null EB-like clusters with specific growth factors caused them to differentiate into cells representative of all three embryonic germ layers, establishing the multipotent nature of CD47-null cells. However, the researchers did not observe the formation of teratomas when CD47-null EB-like clusters were injected into immune-compromised mice, suggesting that the cells may not be fully pluripotent or that lack of CD47 expression induced phagocytic clearance of the cells.
To see whether CD47 regulates similar pathways in vivo, the investigators examined tissues from wild type and CD47-null mice. They found that c-Myc expression was increased in a number of organs, especially the spleen, in mice lacking CD47 compared to wild type animals. Sox2 and Oct4 levels were also elevated in CD47-null tissues. Since the spleen contains a resident population of stem cells, the scientists assessed differences there with and without CD47. While there was no difference in the subcapsular region between wild type and CD47-null tissues, the red pulp from mice lacking CD47 showed extensive Sox2 staining, supporting the idea that loss of CD47 increases the number of stem cells in vivo.
The preceding experiments suggest an association between CD47, c-Myc, and stem cell maintenance. To determine if CD47 directly regulates c-Myc, the researchers treated T cells from c-Myc-EGFP knock-in mice with a CD47-targeted morpholino and observed a seven-fold increase in c-Myc mRNA. Injecting wild type mice with the morpholino decreased CD47 expression and increased c-Myc, Oct4, and Sox2 mRNA in the spleen. In contrast, re-expressing CD47 in CD47-null endothelial cells reduced their proliferation and viability and decreased c-Myc mRNA levels. These results show that CD47 directly inhibits c-Myc expression.
The scientists next asked whether thrombospondin-1, the ligand for CD47, was also capable of regulating c-Myc. Treating human Jurkat T lymphoma cells with thrombospondin-1 strongly inhibited c-Myc expression. However, thrombospondin-1 was not able to reduce c-Myc levels in Jurkat cells lacking CD47. By re-expressing CD47 in these cells, the investigators restored thrombospondin-1 regulation of c-Myc. Thrombospondin-1 also regulated c-Myc levels in vivo. Thrombospondin-1-null mice expressed elevated c-Myc, Oct4, and Sox2 mRNA in the spleen.
Based on these results, elevated CD47 expression should be a disadvantage to cancer cells because of the receptor’s ability to suppress c-Myc and cell proliferation and survival. Yet many cancer cells overexpress CD47 and continue to proliferate rapidly. The researchers hypothesized that dysregulation of c-Myc in some cancers may bypass regulation by CD47. In support of this idea, overexpression of CD47 failed to inhibit growth or induce death in Rat1 fibroblasts, Raji Burkitt lymphoma cells, and B16 melanoma cells. Rat1 cells constitutively express c-Myc-estrogen receptor chimeric proteins, and the c-Myc promoter is known to be altered in Raji cells, suggesting that an intact c-Myc promoter is necessary for CD47-mediated regulation.
Together these studies have revealed CD47 and its ligand, thrombospondin-1, to be potent regulators of cell growth, differentiation, and survival via c-Myc. Though further research is needed, treatments that antagonize CD47 signaling may promote tissue regeneration and recovery from injury. They may also be useful for expanding stem cells ex vivo without the need for viruses or plasmids that overexpress oncogenes. CD47 antagonists may also play a role in cancer treatment by enhancing the resistance of normal cells to radiation therapy without protecting cancer cells that have altered c-Myc regulation.Summary Posted: 04/2013
Kaur S, Soto-Pantoja DR, Stein EV, Liu C, Elkahloun AG, Pendrak ML, Nicolae A, Singh SP, Nie Z, Levens D, Isenberg JS, and Roberts DD. Thrombospondin-1 Signaling through CD47 Inhibits Self-Renewal by Regulating c-Myc and Other Stem Cell Transcription Factors. Scientific Reports. 2013 Apr 17 PubMed Link