Rôle de l’AMPK au cours de l’érythropoïèse murine et humaine

AMPK (AMP-activated protein kinase) is a heterotrimeric complex containing α, β, and γ subunits, known for its role in the maintenance of cellular energy homeostasis. It has been shown that Ampk is involved in maintaining integrity of murine RBCs as well as their survival. Indeed, Ampk α1-/- and Ampk γ1-/- mice present a hemolytic anemia, and their RBCs show elasticity defects in their plasma membrane. We hypothesized that the alterations observed in AMPK-deficient erythroblasts were the results of a lack of Ampk earlier during erythroid differentiation. Therefore, we aim to study the role of AMPK during human and murine erythropoiesis. We showed that the absence or activation of Ampk in mice does not affect either the survival, proliferation, or the differentiation of KO erythroblasts compared to WT ones. In human, our data show that the knockdown of the α1 subunit expression by shRNA induces a slowing down of cell proliferation and a dyserythropoiesis, indicated by the shift in pattern of cell surface markers expression during differentiation. In addition, we showed that phosphorylation of AMPK (Thr172) and its target ACC (Ser 79) are elevated in immature (Pro-E –Baso-E) erythroblasts, and then decreased conjointly with the erythroid differentiation. AMPK activation in immature erythroblasts has no effect. Conversely, in mature (Poly-E – Retic) erythroblasts, the persistence of AMPK expression induces a cell cycle arrest in S phase, followed by the induction of autophagy, and of caspase-dependent apoptosis with a differentiation arrest at basophilic erythroblast stage. Our results demonstrate the importance of finely-tuned regulation of AMPK during adult human erythropoiesis. AMPK is needed for efficient erythropoiesis in human, whereas it is involved solely in RBCs function in mice, showcasing yet another contrasting point between human and mouse erythropoiesis.L’érythropoïèse adulte est un processus complexe qui a lieu dans la moelle osseuse, il aboutit à la formation de globules rouges (GR) à partir des cellules souches hématopoïétiques. L’AMPK (AMP-activated protein kinase) est un complexe hétérotrimérique (αβγ) connu pour son rôle de régulateur du métabolisme énergétique cellulaire. L'implication de l’AMPK dans le maintien de la survie et de l’intégrité des globules rouges murins a été démontrée. En effet, les souris invalidées pour Ampk α1, β1 ou γ1 présentent une anémie hémolytique due à une anomalie de la déformabilité des globules rouges. Nous avons émis l’hypothèse que les altérations observées dans les érythrocytes déficients en AMPK pourraient se mettre en place au cours du processus de différenciation des érythroblastes. L’objectif de ce travail est donc d’étudier le rôle de l'AMPK au cours de l’érythropoïèse murine et humaine adulte. Chez la souris, nos résultats démontrent que l’absence de l’AMPK n’affecte ni la prolifération ni la survie ni la différenciation des érythroblastes Ampkα1-/-. De la même façon, l’activation de l’AMPK n’a pas d’effet sur les érythroblastes murins. Chez l’homme, nous avons montré par une approche shRNA que l’inhibition de l’expression de la sous-unité α1 de l’AMPK induit un ralentissement de la prolifération cellulaire et une anomalie de l’érythropoïèse révélée par une modification de l’expression des protéines membranaires à la surface des érythroblastes au cours de la différenciation. Nous avons également montré que l’AMPK est fortement activée dans les érythroblastes immatures (Pro-E –Baso-E) et que cette activation diminue dans les érythroblastes matures (Poly-E – Retic). Une activation de l’AMPK par des activateurs directs (GSK621 et 991) dans les érythroblastes immatures n’a pas d’effet. Par contre, le maintien de son activation par les activateurs directs dans les érythroblastes matures induit, un arrêt du cycle cellulaire en phase S, une induction de l’autophagie, une apoptose caspase dépendante conduisant à un arrêt de la différenciation au stade Baso-E. Ces résultats montrent l’importance de la diminution de l’activation de l’AMPK pour la survie et la différenciation des érythroblastes matures. L’AMPK est donc importante pour la différenciation des cellules érythroïdes humaines alors que chez la souris, elle est impliquée dans le fonctionnement du globule rouge. Notre travail illustre donc un nouveau point de divergence entre l’érythropoïèse murine et l’érythropoïèse humaine

Rôle de l’AMPK au cours de l’érythropoïèse murine et humaine

L’érythropoïèse adulte est un processus complexe qui a lieu dans la moelle osseuse, il aboutit à la formation de globules rouges (GR) à partir des cellules souches hématopoïétiques. L’AMPK (AMP-activated protein kinase) est un complexe hétérotrimérique (αβγ) connu pour son rôle de régulateur du métabolisme énergétique cellulaire. L'implication de l’AMPK dans le maintien de la survie et de l’intégrité des globules rouges murins a été démontrée. En effet, les souris invalidées pour Ampk α1, β1 ou γ1 présentent une anémie hémolytique due à une anomalie de la déformabilité des globules rouges. Nous avons émis l’hypothèse que les altérations observées dans les érythrocytes déficients en AMPK pourraient se mettre en place au cours du processus de différenciation des érythroblastes. L’objectif de ce travail est donc d’étudier le rôle de l'AMPK au cours de l’érythropoïèse murine et humaine adulte. Chez la souris, nos résultats démontrent que l’absence de l’AMPK n’affecte ni la prolifération ni la survie ni la différenciation des érythroblastes Ampkα1-/-. De la même façon, l’activation de l’AMPK n’a pas d’effet sur les érythroblastes murins. Chez l’homme, nous avons montré par une approche shRNA que l’inhibition de l’expression de la sous-unité α1 de l’AMPK induit un ralentissement de la prolifération cellulaire et une anomalie de l’érythropoïèse révélée par une modification de l’expression des protéines membranaires à la surface des érythroblastes au cours de la différenciation. Nous avons également montré que l’AMPK est fortement activée dans les érythroblastes immatures (Pro-E –Baso-E) et que cette activation diminue dans les érythroblastes matures (Poly-E – Retic). Une activation de l’AMPK par des activateurs directs (GSK621 et 991) dans les érythroblastes immatures n’a pas d’effet. Par contre, le maintien de son activation par les activateurs directs dans les érythroblastes matures induit, un arrêt du cycle cellulaire en phase S, une induction de l’autophagie, une apoptose caspase dépendante conduisant à un arrêt de la différenciation au stade Baso-E. Ces résultats montrent l’importance de la diminution de l’activation de l’AMPK pour la survie et la différenciation des érythroblastes matures. L’AMPK est donc importante pour la différenciation des cellules érythroïdes humaines alors que chez la souris, elle est impliquée dans le fonctionnement du globule rouge. Notre travail illustre donc un nouveau point de divergence entre l’érythropoïèse murine et l’érythropoïèse humaine.AMPK (AMP-activated protein kinase) is a heterotrimeric complex containing α, β, and γ subunits, known for its role in the maintenance of cellular energy homeostasis. It has been shown that Ampk is involved in maintaining integrity of murine RBCs as well as their survival. Indeed, Ampk α1-/- and Ampk γ1-/- mice present a hemolytic anemia, and their RBCs show elasticity defects in their plasma membrane. We hypothesized that the alterations observed in AMPK-deficient erythroblasts were the results of a lack of Ampk earlier during erythroid differentiation. Therefore, we aim to study the role of AMPK during human and murine erythropoiesis. We showed that the absence or activation of Ampk in mice does not affect either the survival, proliferation, or the differentiation of KO erythroblasts compared to WT ones. In human, our data show that the knockdown of the α1 subunit expression by shRNA induces a slowing down of cell proliferation and a dyserythropoiesis, indicated by the shift in pattern of cell surface markers expression during differentiation. In addition, we showed that phosphorylation of AMPK (Thr172) and its target ACC (Ser 79) are elevated in immature (Pro-E –Baso-E) erythroblasts, and then decreased conjointly with the erythroid differentiation. AMPK activation in immature erythroblasts has no effect. Conversely, in mature (Poly-E – Retic) erythroblasts, the persistence of AMPK expression induces a cell cycle arrest in S phase, followed by the induction of autophagy, and of caspase-dependent apoptosis with a differentiation arrest at basophilic erythroblast stage. Our results demonstrate the importance of finely-tuned regulation of AMPK during adult human erythropoiesis. AMPK is needed for efficient erythropoiesis in human, whereas it is involved solely in RBCs function in mice, showcasing yet another contrasting point between human and mouse erythropoiesis

Plasmodium falciparum gametocyte-infected erythrocytes do not adhere to human primary erythroblasts

Abstract Plasmodium falciparum gametocytes, the sexual stages responsible for malaria parasite transmission, develop in the human bone marrow parenchyma in proximity to the erythroblastic islands. Yet, mechanisms underlying gametocytes interactions with these islands are unknown. Here, we have investigated whether gametocyte-infected erythrocytes (GIE) adhere to erythroid precursors, and whether a putative adhesion may be mediated by a mechanism similar to the adhesion of erythrocytes infected with P. falciparum asexual stages to uninfected erythrocytes. Cell-cell adhesion assays with human primary erythroblasts or erythroid cell lines revealed that immature GIE do not specifically adhere to erythroid precursors. To determine whether adhesion may be dependent on binding of STEVOR proteins to Glycophorin C on the surface of erythroid cells, we used clonal lines and transgenic parasites that overexpress specific STEVOR proteins known to bind to Glycophorin C in asexual stages. Our results indicate that GIE overexpressing STEVOR do not specifically adhere to erythroblasts, in agreement with our observation that the STEVOR adhesive domain is not exposed at the surface of GIE

Finely-tuned regulation of AMP-activated protein kinase is crucial for human adult erythropoiesis

International audienceAMP-activated protein kinase (AMPK) is a heterotrimeric complex containing α, β, and γ subunits involved in maintaining integrity and survival of murine red blood cells. Indeed, Ampk α1-/- , Ampk β1-/- and Ampk γ1-/- mice develop hemolytic anemia and the plasma membrane of their red blood cells shows elasticity defects. The membrane composition evolves continuously along erythropoiesis and during red blood cell maturation; defects due to the absence of Ampk could be initiated during erythropoiesis. We, therefore, studied the role of AMPK during human erythropoiesis. Our data show that AMPK activation had two distinct phases in primary erythroblasts. The phosphorylation of AMPK (Thr172) and its target acetyl CoA carboxylase (Ser79) was elevated in immature erythroblasts (glycophorin Alow), then decreased conjointly with erythroid differentiation. In erythroblasts, knockdown of the α1 catalytic subunit by short hairpin RNA led to a decrease in cell proliferation and alterations in the expression of membrane proteins (band 3 and glycophorin A) associated with an increase in phosphorylation of adducin (Ser726). AMPK activation in mature erythroblasts (glycophorin Ahigh), achieved through the use of direct activators (GSK621 and compound 991), induced cell cycle arrest in the S phase, the induction of autophagy and caspase-dependent apoptosis, whereas no such effects were observed in similarly treated immature erythroblasts. Thus, our work suggests that AMPK activation during the final stages of erythropoiesis is deleterious. As the use of direct AMPK activators is being considered as a treatment in several pathologies (diabetes, acute myeloid leukemia), this observation is pivotal. Our data highlighted the importance of the finely-tuned regulation of AMPK during human erythropoiesis

Comprehensive proteomic analysis of murine terminal erythroid differentiation

International audienceMurine-based cellular models have provided and continue to provide many useful insights into the fundamental mechanisms of erythropoiesis, as well as insights into the pathophysiology of inherited and acquired red cell disorders. Although detailed information on many aspects of these cell models is available, comprehensive proteomic data are lacking. This is a critical knowledge gap, as proteins are effectors of most biologic processes. To address this critical unmet need, proteomes of the murine cell lines Friend erythroleukemia (MEL), GATA1 erythroid (G1ER), and embryonic stem cell-derived erythroid progenitor (MEDEP) and proteomes of cultured murine marrow-derived erythroblasts at different stages of terminal erythroid differentiation were analyzed. The proteomes of MEDEP cells and primary murine erythroid cells were most similar, whereas those of MEL and G1ER cells were more distantly related. We demonstrated that the overall cellular content of histones does not decrease during terminal differentiation, despite strong chromatin condensation. Comparison of murine and human proteomes throughout terminal erythroid differentiation revealed that many noted transcriptomic changes were significantly dampened at the proteome level, especially at the end of the terminal differentiation process. Analysis of the early events associated with induction of terminal differentiation in MEDEP cells revealed divergent alterations in associated transcriptomes and proteomes. These proteomic data are powerful and valuable tools for the study of fundamental mechanisms of normal and disordered erythropoiesis and will be of broad interest to a wide range of investigators for making the appropriate choice of various cell lines to study inherited and acquired diseases of the erythrocyte

ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription

International audienceErythropoietin (EPO) is a key regulator of erythropoiesis. The embryonic liver is the main site of erythropoietin synthesis, after which the kidney takes over. The adult liver retains the ability to express EPO, and we discovered here new players of this transcription, distinct from the classical hypoxia-inducible factor pathway. In mice, genetically invalidated in hepatocytes for the chromatin remodeler Arid1a, and for Apc, the major silencer of Wnt pathway, chromatin was more accessible and histone marks turned into active ones at the Epo downstream enhancer. Activating b-catenin signaling increased binding of Tcf4/b-catenin complex and upregulated its enhancer function. The loss of Arid1a together with b-catenin signaling, resulted in cell-autonomous EPO transcription in mouse and human hepatocytes. In mice with Apc-Arid1a gene invalidations in single hepatocytes, Epo de novo synthesis led to its secretion, to splenic erythropoiesis and to dramatic erythrocytosis. Thus, we identified new hepatic EPO regulation mechanism stimulating erythropoiesis