RESUMEN
Thrombospondin-1 is a secreted matricellular glycoprotein that modulates cell behavior by interacting with components of the extracellular matrix and with several cell surface receptors. Its presence in the extracellular matrix is induced by injuries that cause thrombospondin-1 release from platelets and conditions including hyperglycemia, ischemia, and aging that stimulate its expression by many cell types. Conversely, rapid receptor-mediated clearance of thrombospondin-1 from the extracellular space limits its sustained presence in the extracellular space and maintains sub-nanomolar physiological concentrations in blood plasma. Roles for thrombospondin-1 signaling, mediated by specific cellular receptors or by activation of latent TGFß, have been defined in T and B lymphocytes, natural killer cells, macrophages, neutrophils, and dendritic cells. In addition to regulating physiological nitric oxide signaling and responses of cells to stress, studies in mice lacking thrombospondin-1 or its receptors have revealed important roles for thrombospondin-1 in regulating immune responses in infectious and autoimmune diseases and antitumor immunity.
Asunto(s)
Antígeno CD47 , Transducción de Señal , Animales , Ratones , Antígeno CD47/metabolismo , Transducción de Señal/fisiología , Matriz Extracelular/metabolismo , TrombospondinasRESUMEN
Extramedullary erythropoiesis is not expected in healthy adult mice, but erythropoietic gene expression was elevated in lineage-depleted spleen cells from Cd47-/- mice. Expression of several genes associated with early stages of erythropoiesis was elevated in mice lacking CD47 or its signaling ligand thrombospondin-1, consistent with previous evidence that this signaling pathway inhibits expression of multipotent stem cell transcription factors in spleen. In contrast, cells expressing markers of committed erythroid progenitors were more abundant in Cd47-/- spleens but significantly depleted in Thbs1-/- spleens. Single-cell transcriptome and flow cytometry analyses indicated that loss of CD47 is associated with accumulation and increased proliferation in spleen of Ter119-CD34+ progenitors and Ter119+CD34- committed erythroid progenitors with elevated mRNA expression of Kit, Ermap, and Tfrc. Induction of committed erythroid precursors is consistent with the known function of CD47 to limit the phagocytic removal of aged erythrocytes. Conversely, loss of thrombospondin-1 delays the turnover of aged red blood cells, which may account for the suppression of committed erythroid precursors in Thbs1-/- spleens relative to basal levels in wild-type mice. In addition to defining a role for CD47 to limit extramedullary erythropoiesis, these studies reveal a thrombospondin-1-dependent basal level of extramedullary erythropoiesis in adult mouse spleen.
Asunto(s)
Antígeno CD47 , Eritropoyesis , Bazo , Trombospondina 1 , Animales , Antígeno CD47/metabolismo , Antígeno CD47/genética , Trombospondina 1/metabolismo , Trombospondina 1/genética , Bazo/metabolismo , Ratones , Ratones Noqueados , Regulación de la Expresión Génica , Ratones Endogámicos C57BL , Células Precursoras Eritroides/metabolismoRESUMEN
Signal regulatory protein-α (SIRPα, SHPS-1, CD172a) expressed on myeloid cells transmits inhibitory signals when it engages its counter-receptor CD47 on an adjacent cell. Elevated CD47 expression on some cancer cells thereby serves as an innate immune checkpoint that limits phagocytic clearance of tumor cells by macrophages and antigen presentation to T cells. Antibodies and recombinant SIRPα constructs that block the CD47-SIRPα interaction on macrophages exhibit anti-tumor activities in mouse models and are in ongoing clinical trials for treating several human cancers. Based on prior evidence that engaging SIRPα can also alter CD47 signaling in some nonmalignant cells, we compared direct effects of recombinant SIRPα-Fc and a humanized CD47 antibody that inhibits CD47-SIRPα interaction (CC-90002) on CD47 signaling in cancer stem cells derived from the MDA-MB- 231 triple-negative breast carcinoma cell line. Treatment with SIRPα-Fc significantly increased the formation of mammospheres by breast cancer stem cells as compared to CC-90002 treatment or controls. Furthermore, SIRPα-Fc treatment upregulated mRNA and protein expression of ALDH1 and altered the expression of genes involved in epithelial/mesenchymal transition pathways that are associated with a poor prognosis and enhanced metastatic activity. This indicates that SIRPα-Fc has CD47-mediated agonist activities in breast cancer stem cells affecting proliferation and metastasis pathways that differ from those of CC-90002. This SIRPα-induced CD47 signaling in breast carcinoma cells may limit the efficacy of SIRPα decoy therapeutics intended to stimulate innate antitumor immune responses.
RESUMEN
Extracellular vesicles (EVs) released by endothelial cells support vascular homeostasis. To better understand endothelial cell EV biogenesis, we examined cultured human umbilical vein endothelial cells (HUVECs) prepared by rapid freezing, freeze-substitution, and serial thin section electron microscopy (EM). Thin sections of HUVECs revealed clusters of membrane protrusions on the otherwise smooth cell surface. The protrusions contained membrane-bound organelles, including multivesicular bodies (MVBs), and appeared to be on the verge of pinching off to form microvesicles. Beyond cell peripheries, membrane-bound vesicles with internal MVBs were observed, and serial sections confirmed that they were not connected to cells. These observations are consistent with the notion that these multi-compartmented microvesicles (MCMVs) pinch-off from protrusions. Remarkably, omega figures formed by fusion of vesicles with the MCMV limiting membrane were directly observed, apparently releasing exosomes from the MCMV. In summary, MCMVs are a novel form of EV that bud from membrane protrusions on the HUVEC surface, contain MVBs and release exosomes. These observations suggest that exosomes can be harboured within and released from transiting microvesicles after departure from the parent cell, constituting a new site of exosome biogenesis occurring from endothelial and potentially additional cell types.