Lionel Blanc

Exosome secretion, including the DNA damage-induced p53-dependent secretory pathway, is severely compromised in TSAP6/Steap3-null mice

TSAP6 (tumor suppressor-activated pathway 6), also known as Steap3, is a direct p53 transcriptional target gene. It regulates protein secretion, for example translationally controlled tumor protein (TCTP), which is implicated in tumor reversion. In keeping with the latter, we show herein that TSAP6 is a glycosylated protein present in the trans-Golgi network, endosomal–vesicular compartment and cytoplasmic membrane. To further investigate the physiological function of TSAP6, we have generated TSAP6-deficient mice. These mice exhibit microcytic anemia with abnormal reticulocyte maturation and deficient transferrin receptor downregulation, a process known to be dependent on exosomal secretion. Moreover, we provide direct evidence that exosome production is severely compromised in TSAP6-null cells. Finally, we show that the DNA damage-induced p53-dependent nonclassical exosomal secretory pathway is abrogated in TSAP6-null cells. Given the fact that exosomes are used as cell-free vaccines against cancer and that they could be involved in the biogenesis and spread of human immunodeficiency virus, it is important to understand their regulation. The results presented here provide the first genetic demonstration that exosome formation is a tightly controlled biological process dependent of TSAP6.

Galectin-5 is bound onto the surface of rat reticulocyte exosomes and modulates vesicle uptake by macrophages

Reticulocytes release small membrane vesicles termed exosomes during their maturation into erythrocytes. Exosomes are intraluminal vesicles of multivesicular endosomes released into the extracellular medium by fusion of these endosomal compartments with the plasma membrane. This secretion pathway contributes to reticulocyte plasma membrane remodeling by eliminating certain membrane glycoproteins. We show in this study that galectin-5, although mainly cytosolic, is also present on the cell surface of rat reticulocytes and erythrocytes. In addition, in reticulocytes, it resides in the endosomal compartment. We document galectin-5 translocation from the cytosol into the endosome lumen, leading to its secretion in association with exosomes. Galectin-5 bound onto the vesicle surface may function in sorting galactose-bearing glycoconjugates. Fittingly, we found that Lamp2, a major cellular glycoprotein presenting galectin-reactive poly-N-acetylactosamine chains, is lost during reticulocyte maturation. It is associated with released exosomes, suggestive of binding to galectin-5. Finally, we reveal that the uptake of rat reticulocyte exosomes by macrophages is dependent on temperature and the mechanoenzyme dynamin and that exosome uptake is decreased by adding galectin-5. These data imply galectin-5 functionality in the exosomal sorting pathway during rat reticulocyte maturation.

Degradation of AP2 During Reticulocyte Maturation Enhances Binding of Hsc70 and Alix to a Common Site on TfR for Sorting into Exosomes

Reticulocytes release small membrane vesicles termed exosomes during their maturation in erythrocytes. The transferrin receptor (TfR) is completely lost from the red cell surface by its segregation in the secreted vesicles where it interacts with the heat shock cognate 70 kDa protein (hsc70). We have now determined a region of the TfR that can potentially interact with hsc70. The peptide P1 (YTRFSLARQV) from the TfR cytosolic domain: (i) binds to hsc70 (ii) with an increased affinity in oxidative conditions, (iii) competes for binding of an unfolded protein to hsc70, and (iv) inhibits the interaction of hsc70 with a recombinant protein corresponding to the cytosolic domain of the receptor. This peptide encompasses the internalization motif (YTRF) of the receptor, and accordingly an affinity column made with the immobilized peptide retains hsc70 and also the AP2 adaptor complex. On the other hand, we show that AP2 is degraded by the proteasome system during reticulocyte maturation and that the presence of the proteasome inhibitor during in vitro red cell maturation inhibits AP2 degradation and specifically decreases TfR secretion via exosomes. Finally, coimmunoprecipitation of Alix with the exosomal TfR, and binding of P1 peptide to the Alix homolog PalA suggest that Alix also interacts with the YTRF motif and contributes to exosomal TfR sorting.

The Glut1 and Glut4 glucose transporters are differentially expressed during perinatal and postnatal erythropoiesis

Glucose is a major source of energy for living organisms, and its transport in vertebrates is a universally conserved property. Of all cell lineages, human erythrocytes express the highest level of the Glut1 glucose transporter with more than 200,000 molecules per cell. However, we recently reported that erythrocyte Glut1 expression is a specific trait of vitamin C-deficient mammalian species, comprising only higher primates, guinea pigs, and fruit bats. Here, we show that in all other tested mammalian species, Glut1 was transiently expressed in erythrocytes during the neonatal period. Glut1 was up-regulated during the erythroblast stage of erythroid differentiation and was present on the vast majority of murine red blood cells (RBCs) at birth. Notably though, Glut1 was not induced in adult mice undergoing anemia-induced erythropoiesis, and under these conditions, the up-regulation of a distinct transporter, Glut4, was responsible for an increased glucose transport. Sp3 and Sp1 transcriptions factors have been proposed to regulate Glut1 transcription, and we find that the concomitant repression of Glut1 and induction of Glut4 was associated with a significantly augmented Sp3/Sp1 ratio. Glucose transporter expression patterns in mice and human erythrocytes are therefore distinct. In mice, there is a postnatal switch from Glut1 to Glut4, with Glut4 further up-regulated under anemic conditions.

Reticulocyte membrane remodeling: contribution of the exosome pathway.

Purpose of reviewIn addition to the loss of all the internal compartments, reticulocyte maturation is characterized by an extensive membrane remodeling. Exosomal secretion contributes to this process by eliminating specific proteins. Recent findingsRegulation in the exosomal sorting of the water channel aquaporin-1 represents a newly described mechanism by which reticulocytes could adapt to environmental modifications. The extracellular osmotic conditions found in the peripheral circulation vs. the bone marrow could dictate a lower level of expression of aquaporin-1 on the mature red cell surface. In addition, a new mechanism for protein sorting to exosomes, involving an endogenous lectin, has been pinpointed in rat reticulocytes. Galectin-5 is secreted by a so-called alternative pathway, and could be involved in the sorting of galactoside-bearing glycoconjugates, since it was found associated with the surface of released exosomes. SummarySecretion of exosomes during reticulocyte maturation is an integral part of the red cell differentiation program and illustrates specific mechanisms in terms of biogenesis, protein sorting and fate, which are far from completely understood and open new paths for research in the red cell physiology field.

The water channel aquaporin-1 partitions into exosomes during reticulocyte maturation: implication for the regulation of cell volume

Aquaporin-1 (AQP-1), the universal water channel, is responsible for rapid response of cell volume to changes in plasma tonicity. In the membrane of the red cell the concentration of the protein is tightly controlled. Here, we show that AQP-1 is partially lost during in vitro maturation of mouse reticulocytes and that it is associated with exosomes, released throughout this process. AQP-1 in young reticulocytes localizes to the plasma membrane and also in endosomal compartments and exosomes, formed both in vitro and in vivo. During maturation a part of the total pool of AQP-1 is differentially sorted and released via the exosomal pathway. A proteasome inhibitor, MG132, suppresses secretion of AQP-1, implying that ubiquitination is a sorting signal for its release. We further show that modulation of medium tonicity in vitro regulates the secretion of AQP-1, thus showing that extracellular osmotic conditions can drive sorting of selected proteins by the exosomal pathway. These results lead us to suggest that AQP-1 sorting into exosomes may be the mechanism by which the reticulocyte adapts to environmental changes during its maturation.

Diamond Blackfan anemia: a model for the translational approach to understanding human disease

Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome. As with the other rare inherited bone marrow failure syndromes, the study of these disorders provides important insights into basic biology and, in the case of DBA, ribosome biology; the disruption of which characterizes the disorder. Thus DBA serves as a paradigm for translational medicine in which the efforts of clinicians to manage DBA have informed laboratory scientists who, in turn, have stimulated clinical researchers to utilize scientific discovery to provide improved care. In this review we describe the clinical syndrome Diamond Blackfan anemia and, in particular, we demonstrate how the study of DBA has allowed scientific inquiry to create opportunities for progress in its understanding and treatment.

HMGB1 mediates splenomegaly and expansion of splenic CD11b+ Ly‐6Chigh inflammatory monocytes in murine sepsis survivors

More than 500,000 hospitalized patients survive severe sepsis annually in the USA. Recent epidemiological evidence, however, demonstrated that these survivors have significant morbidity and mortality, with 3‐year fatality rates higher than 70%. To investigate the mechanisms underlying persistent functional impairment in sepsis survivors, here we developed a model to study severe sepsis survivors following cecal ligation and puncture (CLP).

Pomalidomide reverses γ-globin silencing through the transcriptional reprogramming of adult hematopoietic progenitors

Current therapeutic strategies for sickle cell anemia are aimed at reactivating fetal hemoglobin. Pomalidomide, a third-generation immunomodulatory drug, was proposed to induce fetal hemoglobin production by an unknown mechanism. Here, we report that pomalidomide induced a fetal-like erythroid differentiation program, leading to a reversion of γ-globin silencing in adult human erythroblasts. Pomalidomide acted early by transiently delaying erythropoiesis at the burst-forming unit-erythroid/colony-forming unit-erythroid transition, but without affecting terminal differentiation. Further, the transcription networks involved in γ-globin repression were selectively and differentially affected by pomalidomide including BCL11A, SOX6, IKZF1, KLF1, and LSD1. IKAROS (IKZF1), a known target of pomalidomide, was degraded by the proteasome, but was not the key effector of this program, because genetic ablation of IKZF1 did not phenocopy pomalidomide treatment. Notably, the pomalidomide-induced reprogramming was conserved in hematopoietic progenitors from individuals with sickle cell anemia. Moreover, multiple myeloma patients treated with pomalidomide demonstrated increased in vivo γ-globin levels in their erythrocytes. Together, these data reveal the molecular mechanisms by which pomalidomide reactivates fetal hemoglobin, reinforcing its potential as a treatment for patients with β-hemoglobinopathies.

Control of Erythrocyte Membrane-Skeletal Cohesion by the Spectrin-Membrane Linkage

Spectrin tetramer is the major structural member of the membrane-associated skeletal network of red cells. We show here that disruption of the spectrin-ankyrin-band 3 link to the membrane leads to dissociation of a large proportion of the tetramers into dimers. Noncovalent perturbation of the linkage was induced by a peptide containing the ankyrin-binding site of the spectrin beta-chain, and covalent perturbation by treatment with the thiol reagent, N-ethylmaleimide (NEM). This reagent left the intrinsic self-association capacity of the spectrin dimers unaffected and disturbed only the ankyrin-band 3 interaction. The dissociation of spectrin tetramers on the membrane into functional dimers was confirmed by the binding of a spectrin peptide directed against the self-association sites. Dissociation of the tetramers resulted, we infer, from detachment of the proximal ends of the constituent dimers from the membrane, thereby reducing their proximity to one another and thus weakening their association. The measured affinity of the interaction of the peptides with the free dimer ends on the membrane permits an estimate of the equilibrium between intact and dissociated tetramers on the native membrane. This indicates that in the physiological state the equilibrium proportion of the dissociated tetramers may be as high as 5-10%. These findings enabled us to identify an additional important functional role for the spectrin-ankyrin-band 3 link in regulating spectrin self-association in the red cell membrane.